Alpha (1,2) fucosyltransferase syngenes for use in the production of fucosylated oligosaccharides

ABSTRACT

The invention provides compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.

RELATED APPLICATIONS

This application is a national stage application, filed under 35 U.S.C. § 371, of PCT International Patent Application No. PCT/US2015/030823, filed on May 14, 2015, and claims benefit of priority to U.S. Provisional Patent Application No. 61/993,742, filed on May 15, 2014, both of which, including their contents, are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web. The content of the text file named “37847-517001US_ST25.txt”, which was created on Oct. 31, 2016 and is 786 KB in size, is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention provides compositions and methods for producing purified oligosaccharides, in particular certain fucosylated oligosaccharides that are typically found in human milk.

BACKGROUND OF THE INVENTION

Human milk contains a diverse and abundant set of neutral and acidic oligosaccharides. More than 130 different complex oligosaccharides have been identified in human milk, and their structural diversity and abundance is unique to humans. Although these molecules may not be utilized directly by infants for nutrition, they nevertheless serve critical roles in the establishment of a healthy gut microbiome, in the prevention of disease, and in immune function. Prior to the invention described herein, the ability to produce human milk oligosaccharides (HMOS) inexpensively was problematic. For example, their production through chemical synthesis was limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost. As such, there is a pressing need for new strategies to inexpensively manufacture large quantities of HMOS.

SUMMARY OF THE INVENTION

The invention features an efficient and economical method for producing fucosylated oligosaccharides. Such production of a fucosylated oligosaccharide is accomplished using an isolated nucleic acid comprising a sequence encoding a lactose-utilizing α (1,2) fucosyltransferase gene product (e.g., polypeptide or protein), which is operably linked to one or more heterologous control sequences that direct the production of the recombinant fucosyltransferase gene product in a host production bacterium such as Escherichia coli (E. coli).

The present disclosure provides novel α (1,2) fucosyltransferases (also referred to herein as α(1,2) FTs) that utilize lactose and catalyzes the transfer of an L-fucose sugar from a GDP-fucose donor substrate to an acceptor substrate in an alpha-1,2-linkage. In a preferred embodiment, the acceptor substrate is an oligosaccharide. The α(1,2) fucosyltransferases identified and described herein are useful for expressing in host bacterium for the production of human milk oligosaccharides (HMOS), such as fucosylated oligosaccharides. Exemplary fucosylated oligosaccharides produced by the methods described herein include 2′-fucosyllactose (2′FL), lactodifucotetraose (LDFT), lacto-N-fucopentaose I (LNF I), or lacto-N-difucohexaose I (LDFH I). The “α(1,2) fucosyltransferases” disclosed herein encompasses the amino acid sequences of the α(1,2) fucosyltransferases and the nucleic acid sequences that encode the α(1,2) fucosyltransferases, as well as variants and fragments thereof that exhibit α(1,2) fucosyltransferase activity. Also within the invention is a nucleic acid construct comprising an isolated nucleic acid encoding a lactose-accepting α (1,2) fucosyltransferase enzyme, said nucleic acid being optionally operably linked to one or more heterologous control sequences that direct the production of the enzyme in a host bacteria production strain.

The amino acid sequence of the lactose-accepting α(1,2) fucosyltransferases described herein is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to Helicobacter pylori 26695 alpha-(1,2) fucosyltransferase (futC or SEQ ID NO: 1). Preferably, the lactose-accepting α(1,2) fucosyltransferases described herein is at least 22% identical to H. pylori FutC, or SEQ ID NO: 1.

In another aspect, the amino acid sequence of the lactose-accepting α(1,2) fucosyltransferases described herein is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to Bacteroides vulgatus alpha-(1,2) fucosyltransferase (FutN or SEQ ID NO: 3). Preferably, the lactose-accepting α(1,2) fucosyltransferases described herein is at least 25% identical to B. vlugatos FutN, or SEQ ID NO: 3.

Alternatively, the exogenous α (1,2) fucosyltransferase preferably comprises at least at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to any one of the novel α (1,2) fucosyltransferases disclosed herein, for example, to the amino acid sequences in Table 1.

Exemplary α(1,2) fucosyltransferases include, but are not limited to, Prevotella melaninogenica FutO, Clostridium bolteae FutP, Clostridium bolteae+13 FutP, Lachnospiraceae sp. FutQ, Methanosphaerula palustris FutR, Tannerella sp. FutS, Bacteroides caccae FutU, Butyrivibrio FutV, Prevotella sp. FutW, Parabacteroides johnsonii FutX, Akkermansia muciniphilia FutY, Salmonella enterica FutZ, Bacteroides sp. FutZA. For example, the α(1,2) fucosyltransferases comprise the amino acid sequences comprising any one of the following: Prevotella melaninogenica FutO (SEQ ID NO: 10), Clostridium bolteae FutP (SEQ ID NO: 11), Clostridium bolteae+13 FutP (SEQ ID NO: 292), Lachnospiraceae sp. FutQ (SEQ ID NO: 12), Methanosphaerula palustris FutR (SEQ ID NO: 13), Tannerella sp. FutS (SEQ ID NO: 14), Bacteroides caccae FutU (SEQ ID NO: 15), Butyrivibrio FutV (SEQ ID NO: 16), Prevotella sp. FutW (SEQ ID NO: 17), Parabacteroides johnsonii FutX (SEQ ID NO: 18), Akkermansia muciniphilia FutY (SEQ ID NO: 19), Salmonella enterica FutZ (SEQ ID NO: 20), and Bacteroides sp. FutZA (SEQ ID NO: 21), or a functional variant or fragment thereof. Other exemplary α(1,2) fucosyltransferases include any of the enzymes listed in Table 1, or functional variants or fragments thereof.

The present invention features a method for producing a fucosylated oligosaccharide in a bacterium by providing bacterium that express at least one exogenous lactose-utilizing α(1,2) fucosyltransferase. The amino acid sequence of the exogenous lactose-utilizing α(1,2) fucosyltransferase is preferably at least 22% identical to H. pylori FutC or at least 25% identical to B. vulgatus FutN. In one aspect, the bacterium also expresses one or more exogenous lactose-utilizing α(1,3) fucosyltransferase enzymes and/or one or more exogenous lactose-utilizing α(1,4) fucosyltransferase enzymes. The combination of fucosyltransferases expressed in the production bacterium is dependent upon the desired fucosylated oligosaccharide product. The method disclosed herein further includes retrieving the fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.

Examples of suitable α(1,3) fucosyltransferase enzymes include, but are not limited to Helicobacter pylori 26695 futA gene (GenBank Accession Number HV532291 (GI:365791177), incorporated herein by reference), H. hepaticus Hh0072, H. pylori 11639 FucT, and H. pylori UA948 FucTa (e.g., GenBank Accession Number AF194963 (GI:28436396), incorporated herein by reference) (Rasko, D. A., Wang, G., Palcic, M. M. & Taylor, D. E. J Biol Chem 275, 4988-4994 (2000)). Examples of suitable α(1,4) fucosyltransferase enzymes include, but are not limited to H. pylori UA948 FucTa (which has has relaxed acceptor specificity and is able to generate both α(1,3)- and α(1,4)-fucosyl linkages). An example of an enzyme possessing only α(1,4) fucosyltransferase activity is given by the FucT III enzyme from Helicobacter pylori strain DMS6709 (e.g., GenBank Accession Number AY450598.1 (GI:40646733), incorporated herein by reference) (S. Rabbani, V. Miksa, B. Wipf, B. Ernst, Glycobiology 15, 1076-83 (2005).)

The invention also features a nucleic acid construct or a vector comprising a nucleic acid enconding at least one α (1,2) fucosyltransferase or variant, or fragment thereof, as described herein. The vector can further include one or more regulatory elements, e.g., a heterologous promoter. By “heterologous” is meant that the control sequence and protein-encoding sequence originate from different bacterial strains. The regulatory elements can be operably linked to a gene encoding a protein, a gene construct encoding a fusion protein gene, or a series of genes linked in an operon in order to express the fusion protein. In yet another aspect, the invention comprises an isolated recombinant cell, e.g., a bacterial cell containing an aforementioned nucleic acid molecule or vector. The nucleic acid is optionally integrated into the genome of the host bacterium. In some embodiments, the nucleic acid construct also further comprises one or more α(1,3) fucosyltransferases and/or α(1,4) fucosyltransferases. Alternatively, the α (1,2) fucosyltransferase also exhibits α(1,3) fucosyltransferase and/or α(1,4) fucosyltransferase activity.

The bacterium utilized in the production methods described herein is genetically engineered to increase the efficiency and yield of fucosylated oligosaccharide products. For example, the host production bacterium is characterized as having a reduced level of β-galactosidase activity, a defective colanic acid synthesis pathway, an inactivated ATP-dependent intracellular protease, an inactivated lacA, or a combination thereof. In one embodiment, the bacterium is characterized as having a reduced level of β-galactosidase activity, a defective colanic acid synthesis pathway, an inactivated ATP-dependent intracellular protease, and an inactivated lacA.

As used herein, an “inactivated” or “inactivation of a” gene, encoded gene product (i.e., polypeptide), or pathway refers to reducing or eliminating the expression (i.e., transcription or translation), protein level (i.e., translation, rate of degradation), or enzymatic activity of the gene, gene product, or pathway. In the instance where a pathway is inactivated, preferably one enzyme or polypeptide in the pathway exhibits reduced or negligible activity. For example, the enzyme in the pathway is altered, deleted or mutated such that the product of the pathway is produced at low levels compared to a wild-type bacterium or an intact pathway. Alternatively, the product of the pathway is not produced. Inactivation of a gene is achieved by deletion or mutation of the gene or regulatory elements of the gene such that the gene is no longer transcribed or translated. Inactivation of a polypeptide can be achieved by deletion or mutation of the gene that encodes the gene product or mutation of the polypeptide to disrupt its activity. Inactivating mutations include additions, deletions or substitutions of one or more nucleotides or amino acids of a nucleic acid or amino acid sequence that results in the reduction or elimination of the expression or activity of the gene or polypeptide. In other embodiments, inactivation of a polypeptide is achieved through the addition of exogenous sequences (i.e., tags) to the N or C-terminus of the polypeptide such that the activity of the polypeptide is reduced or eliminated (i.e., by steric hindrance).

A host bacterium suitable for the production systems described herein exhibits an enhanced or increased cytoplasmic or intracellular pool of lactose and/or GDP-fucose. For example, the bacterium is E. coli and endogenous E. coli metabolic pathways and genes are manipulated in ways that result in the generation of increased cytoplasmic concentrations of lactose and/or GDP-fucose, as compared to levels found in wild type E. coli. Preferably, the bacterium accumulates an increased intracellular lactose pool and an increased intracellular GDP-fucose pool. For example, the bacteria contain at least 10%, 20%, 50%, or 2×, 5×, 10× or more of the levels of intracellular lactose and/or intracellular GDP-fucose compared to a corresponding wild type bacteria that lacks the genetic modifications described herein.

Increased intracellular concentration of lactose in the host bacterium compared to wild-type bacterium is achieved by manipulation of genes and pathways involved in lactose import, export and catabolism. In particular, described herein are methods of increasing intracellular lactose levels in E. coli genetically engineered to produce a human milk oligosaccharide by simultaneous deletion of the endogenous β-galactosidase gene (lacZ) and the lactose operon repressor gene (lad). During construction of this deletion, the lacIq promoter is placed immediately upstream of (contiguous with) the lactose permease gene, lacY, i.e., the sequence of the lacIq promoter is directly upstream and adjacent to the start of the sequence encoding the lacY gene, such that the lacY gene is under transcriptional regulation by the lacIq promoter. The modified strain maintains its ability to transport lactose from the culture medium (via LacY), but is deleted for the wild-type chromosomal copy of the lacZ (encoding β-galactosidase) gene responsible for lactose catabolism. Thus, an intracellular lactose pool is created when the modified strain is cultured in the presence of exogenous lactose.

Another method for increasing the intracellular concentration of lactose in E. coli involves inactivation of the lacA gene. A inactivating mutation, null mutation, or deletion of lacA prevents the formation of intracellular acetyl-lactose, which not only removes this molecule as a contaminant from subsequent purifications, but also eliminates E. coli's ability to export excess lactose from its cytoplasm (Danchin A. Cells need safety valves. Bioessays 2009, July; 31(7):769-73.), thus greatly facilitating purposeful manipulations of the E. coli intracellular lactose pool.

The invention also provides methods for increasing intracellular levels of GDP-fucose in a bacterium by manipulating the organism's endogenous colanic acid biosynthesis pathway. This increase is achieved through a number of genetic modifications of endogenous E. coli genes involved either directly in colanic acid precursor biosynthesis, or in overall control of the colanic acid synthetic regulon. Particularly preferred is inactivation of the genes or encoded polypeptides that act in the colanic acid synthesis pathway after the production of GDP-fucose (the donor substrate) and before the generation of colanic acid. Exemplary colanic acid synthesis genes include, but are not limited to: a wcaJ gene, (e.g., GenBank Accession Number (amino acid) BAA15900 (GI:1736749), incorporated herein by reference), a wcaA gene (e.g., GenBank Accession Number (amino acid) BAA15912.1 (GI:1736762), incorporated herein by reference), a wcaC gene (e.g., GenBank Accession Number (amino acid) BAE76574.1 (GI:85675203), incorporated herein by reference), a wcaE gene (e.g., GenBank Accession Number (amino acid) BAE76572.1 (GI:85675201), incorporated herein by reference), a wcaI gene (e.g., GenBank Accession Number (amino acid) BAA15906.1 (GI:1736756), incorporated herein by reference), a wcaL gene (e.g., GenBank Accession Number (amino acid) BAA15898.1 (GI:1736747), incorporated herein by reference), a wcaB gene (e.g., GenBank Accession Number (amino acid) BAA15911.1 (GI:1736761), incorporated herein by reference), a wcaF gene (e.g., GenBank Accession Number (amino acid) BAA15910.1 (GI:1736760), incorporated herein by reference), a wzxE gene (e.g., GenBank Accession Number (amino acid) BAE77506.1 (GI:85676256), incorporated herein by reference), a wzxC gene, (e.g., GenBank Accession Number (amino acid) BAA15899 (GI:1736748), incorporated herein by reference), a wcaD gene, (e.g., GenBank Accession Number (amino acid) BAE76573 (GI:85675202), incorporated herein by reference), a wza gene (e.g., GenBank Accession Number (amino acid) BAE76576 (GI:85675205), incorporated herein by reference), a wzb gene (e.g., GenBank Accession Number (amino acid) BAE76575 (GI:85675204), incorporated herein by reference), and a wzc gene (e.g., GenBank Accession Number (amino acid) BAA15913 (GI:1736763), incorporated herein by reference).

Preferably, a host bacterium, such as E. coli, is genetically engineered to produce a human milk oligosaccharide by the inactivation of the wcaJ gene, which encoding the UDP-glucose lipid carrier transferase. The inactivation of the wcaJ gene can be by deletion of the gene, a null mutation, or inactivating mutation of the wad gene, such that the activity of the encoded wcaJ is reduced or eliminated compared to wild-type E. coli. In a wcaJ null background, GDP-fucose accumulates in the E. coli cytoplasm.

Over-expression of a positive regulator protein, RcsA (e.g., GenBank Accession Number M58003 (GI:1103316), incorporated herein by reference), in the colanic acid synthesis pathway results in an increase in intracellular GDP-fucose levels. Over-expression of an additional positive regulator of colanic acid biosynthesis, namely RcsB (e.g., GenBank Accession Number E04821 (GI:2173017), incorporated herein by reference), is also utilized, either instead of or in addition to over-expression of RcsA, to increase intracellular GDP-fucose levels.

Alternatively, colanic acid biosynthesis is increased following the introduction of a mutation into the E. coli lon gene (e.g., GenBank Accession Number L20572 (GI:304907), incorporated herein by reference). Lon is an adenosine-5′-triphosphate (ATP)-dependant intracellular protease that is responsible for degrading RcsA, mentioned above as a positive transcriptional regulator of colanic acid biosynthesis in E. coli. In a Ion null background, RcsA is stabilized, RcsA levels increase, the genes responsible for GDP-fucose synthesis in E. coli are up-regulated, and intracellular GDP-fucose concentrations are enhanced. Mutations in Ion suitable for use with the methods presented herein include null mutations or insertions that disrupt the expression or function of lon.

A functional lactose permease gene is also present in the bacterium. The lactose permease gene is an endogenous lactose permease gene or an exogenous lactose permease gene. For example, the lactose permease gene comprises an E. coli lacY gene (e.g., GenBank Accession Number V00295 (GI:41897), incorporated herein by reference). Many bacteria possess the inherent ability to transport lactose from the growth medium into the cell, by utilizing a transport protein that is either a homolog of the E. coli lactose permease (e.g., as found in Bacillus licheniformis), or a transporter that is a member of the ubiquitous PTS sugar transport family (e.g., as found in Lactobacillus casei and Lactobacillus rhamnosus). For bacteria lacking an inherent ability to transport extracellular lactose into the cell cytoplasm, this ability is conferred by an exogenous lactose transporter gene (e.g., E. coli lacY) provided on recombinant DNA constructs, and supplied either on a plasmid expression vector or as exogenous genes integrated into the host chromosome.

As described herein, in some embodiments, the host bacterium preferably has a reduced level of β-galactosidase activity. In the embodiment in which the bacterium is characterized by the deletion of the endogenous β-galactosidase gene, an exogenous β-galactosidase gene is introduced to the bacterium. For example, a plasmid expressing an exogenous β-galactosidase gene is introduced to the bacterium, or recombined or integrated into the host genome. For example, the exogenous β-galactosidase gene is inserted into a gene that is inactivated in the host bacterium, such as the Ion gene.

The exogenous b-galactosidase gene is a functional b-galactosidase gene characterized by a reduced or low level of b-galactosidase activity compared to β-galactosidase activity in wild-type bacteria lacking any genetic manipulation. Exemplary β-galactosidase genes include E. coli lacZ and β-galactosidase genes from any of a number of other organisms (e.g., the lac4 gene of Kluyveromyces lactis (e.g., GenBank Accession Number M84410 (GI:173304), incorporated herein by reference) that catalyzes the hydrolysis of b-galactosides into monosaccharides. The level of β-galactosidase activity in wild-type E. coli bacteria is, for example, 6,000 units. Thus, the reduced β-galactosidase activity level encompassed by engineered host bacterium of the present invention includes less than 6,000 units, less than 5,000 units, less than 4,000 units, less than 3,000 units, less than 2,000 units, less than 1,000 units, less than 900 units, less than 800 units, less than 700 units, less than 600 units, less than 500 units, less than 400 units, less than 300 units, less than 200 units, less than 100 units, or less than 50 units. Low, functional levels of β-galactosidase include β-galactosidase activity levels of between 0.05 and 1,000 units, e.g., between 0.05 and 750 units, between 0.05 and 500 units, between 0.05 and 400 units, between 0.05 and 300 units, between 0.05 and 200 units, between 0.05 and 100 units, between 0.05 and 50 units, between 0.05 and 10 units, between 0.05 and 5 units, between 0.05 and 4 units, between 0.05 and 3 units, or between 0.05 and 2 units of β-galactosidase activity. For unit definition and assays for determining β-galactosidase activity, see Miller J H, Laboratory CSH. Experiments in molecular genetics. Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y.; 1972; (incorporated herein by reference). This low level of cytoplasmic β-galactosidase activity is not high enough to significantly diminish the intracellular lactose pool. The low level of β-galactosidase activity is very useful for the facile removal of undesired residual lactose at the end of fermentations.

Optionally, the bacterium has an inactivated thyA gene. Preferably, a mutation in a thyA gene in the host bacterium allows for the maintenance of plasmids that carry thyA as a selectable marker gene. Exemplary alternative selectable markers include antibiotic resistance genes such as BLA (beta-lactamase), or proBA genes (to complement a proAB host strain proline auxotropy) or purA (to complement a purA host strain adenine auxotrophy).

In one aspect, the E. coli bacterium comprises the genotype ΔampC::P_(trp) ^(B)cI, Δ(lacI-lacZ)::FRT, P_(lacIq)lacY⁺, ΔwcaJ::FRT, thyA::Tn10, Δlon:(npt3, lacZ⁺), ΔlacA, and also comprises any one of the exogenous α(1,2) fucosyltransferases described herein.

The bacterium comprising these characteristics is cultured in the presence of lactose. In some cases, the method further comprises culturing the bacterium in the presence of tryptophan and in the absence of thymidine. The fucosylated oligosaccharide is retrieved from the bacterium (i.e., a cell lysate) or from a culture supernatant of the bacterium.

The invention provides a purified fucosylated oligosaccharide produced by the methods described herein. The fucosylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacterium is used directly in such products. The fucosylated oligosaccharide produced by the engineered bacterium is 2′-fucosyllactose (2′-FL) or lactodifucotetraose (LDFT). The new alpha 1,2-fucosyltransferases are also useful to synthesize HMOS of larger molecular weight bearing alpha 1,2 fucose moieties, e.g., lacto-N-fucopentaose (LNF I) and lacto-N-difucohexaose (LDFH I). For example, to produce LDFT, the host bacterium is engineered to express an exogenous α (1,2) fucosyltransferase that also possesses α (1,3) fucosyltransferase activity, or an exogenous α (1,2) fucosyltransferase and an exogenous α (1,3) fucosyltransferase. For the production of LNF I and LDFH I, the host bacterium is engineered to express an exogenous α (1,2) fucosyltransferase that also possesses α (1,3) fucosyltransferase activity and/or α (1,4) fucosyltransferase activity, or an exogenous α (1,2) fucosyltransferase, an exogenous α (1,3) fucosyltransferas, and an exogenous α (1,4) fucosyltransferase.

A purified fucosylated oligosaccharide produced by the methods described above is also within the invention. The purified oligosaccharide (2′-FL) obtained at the end of the process is a white/slightly off-white, crystalline, sweet powder. For example, an engineered bacterium, bacterial culture supernatant, or bacterial cell lysate according to the invention comprises 2′-FL, LDFT, LNF I or LDFH I produced by the methods described herein, and does not substantially comprise a other fucosylated oligosaccharides prior to purification of the fucosylated oligosaccharide products from the cell, culture supernatant, or lysate. As a general matter, the fucosylated oligosaccharide produced by the methods contains a negligible amount of 3-FL in a 2′-FL-containing cell, cell lysate or culture, or supernatant, e.g., less than 1% of the level of 2′-FL or 0.5% of the level of 2′-FL. Moreover, the fucosylated oligosaccharide produced by the methods described herein also have a minimal amount of contaminating lactose, which can often be co-purified with the fucosylated oligosaccharide product, such as 2′FL. This reduction in contaminating lactose results from the reduced level of β-galactosidase activity present in the engineered host bacterium.

A purified oligosaccharide, e.g., 2′-FL, LDFT, LNF I, or LDFH I, is one that is at least 90%, 95%, 98%, 99%, or 100% (w/w) of the desired oligosaccharide by weight. Purity is assessed by any known method, e.g., thin layer chromatography or other chromatographic techniques known in the art. The invention includes a method of purifying a fucosylated oligosaccharide produced by the genetically engineered bacterium described above, which method comprises separating the desired fucosylated oligosaccharide (e.g., 2′-FL) from contaminants in a bacterial cell lysate or bacterial cell culture supernatant of the bacterium.

The oligosaccharides are purified and used in a number of products for consumption by humans as well as animals, such as companion animals (dogs, cats) as well as livestock (bovine, equine, ovine, caprine, or porcine animals, as well as poultry). For example, a pharmaceutical composition comprises purified 2′-FL and a pharmaceutically-acceptable excipient that is suitable for oral administration. Large quantities of 2′-FL are produced in bacterial hosts, e.g., an E. coli bacterium comprising an exogenous α (1,2) fucosyltransferase gene.

A method of producing a pharmaceutical composition comprising a purified human milk oligosaccharide (HMOS) is carried out by culturing the bacterium described above, purifying the HMOS produced by the bacterium, and combining the HMOS with an excipient or carrier to yield a dietary supplement for oral administration. These compositions are useful in methods of preventing or treating enteric and/or respiratory diseases in infants and adults. Accordingly, the compositions are administered to a subject suffering from or at risk of developing such a disease.

The invention also provides methods of identifying an α (1,2) fucosyltransferase gene capable of synthesizing fucosylated oligosaccharides in a host bacterium, i.e., 2′-fucosyllactose (2′-FL) in E. coli. The method of identifying novel lactose-utilizing, α(1,2)fucosyltransferase enzyme comprises the following steps:

1) performing a computational search of sequence databases to define a broad group of simple sequence homologs of any known, lactose-utilizing α(1,2)fucosyltransferase;

2) using the list from step (1), deriving a search profile containing common sequence and/or structural motifs shared by the members of the list;

3) searching sequence databases, using a derived search profile based on the common sequence or structural motif from step (2) as query, and identifying a candidate sequences, wherein a sequence homology to a reference lactose-utilizing α(1,2)fucosyltransferase is a predetermined percentage threshold;

4) compiling a list of candidate organisms, said organisms being characterized as expressing α(1,2)fucosyl-glycans in a naturally-occurring state;

5) selecting candidate sequences that are derived from candidate organisms to generate a list of candidate lactose-utilizing enzymes;

6) expressing the candidate lactose-utilizing enzyme in a host organism; and

7) testing for lactose-utilizing α(1,2)fucosyltransferase activity, wherein detection of the desired fucosylated oligosaccharide product in said organism indicates that the candidate sequence comprises a novel lactose-utilizing α(1,2)fucosyltransferase. In another embodiment, the search profile is generated from a multiple sequence alignment of the amino acid sequences of more than one enzyme with known α(1,2)fucosyltransferase activity. The database search can then be designed to refine and iteratively search for novel α(1,2)fucosyltransferases with significant sequence similarity to the multiple sequence alignment query.

The invention provides a method of treating, preventing, or reducing the risk of infection in a subject comprising administering to said subject a composition comprising a purified recombinant human milk oligosaccharide, wherein the HMOS binds to a pathogen and wherein the subject is infected with or at risk of infection with the pathogen. In one aspect, the infection is caused by a Norwalk-like virus or Campylobacter jejuni. The subject is preferably a mammal in need of such treatment. The mammal is, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, or a pig. In a preferred embodiment, the mammal is a human. For example, the compositions are formulated into animal feed (e.g., pellets, kibble, mash) or animal food supplements for companion animals, e.g., dogs or cats, as well as livestock or animals grown for food consumption, e.g., cattle, sheep, pigs, chickens, and goats. Preferably, the purified HMOS is formulated into a powder (e.g., infant formula powder or adult nutritional supplement powder, each of which is mixed with a liquid such as water or juice prior to consumption) or in the form of tablets, capsules or pastes or is incorporated as a component in dairy products such as milk, cream, cheese, yogurt or kefir, or as a component in any beverage, or combined in a preparation containing live microbial cultures intended to serve as probiotics, or in prebiotic preparations to enhance the growth of beneficial microorganisms either in vitro or in vivo.

Polynucleotides, polypeptides, and oligosaccharides of the invention are purified and/or isolated. Purified defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. Specifically, as used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, protein or oligosaccharide, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. For example, purified HMOS compositions are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. For example, a “purified protein” refers to a protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. Preferably, the protein constitutes at least 10, 20, 50, 70, 80, 90, 95, 99-100% by dry weight of the purified preparation.

Similarly, by “substantially pure” is meant an oligosaccharide that has been separated from the components that naturally accompany it. Typically, the oligosaccharide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.

By “isolated nucleic acid” is meant a nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones.

A “heterologous promoter” is a promoter which is different from the promoter to which a gene or nucleic acid sequence is operably linked in nature.

The term “overexpress” or “overexpression” refers to a situation in which more factor is expressed by a genetically-altered cell than would be, under the same conditions, by a wild type cell. Similarly, if an unaltered cell does not express a factor that it is genetically altered to produce, the term “express” (as distinguished from “overexpress”) is used indicating the wild type cell did not express the factor at all prior to genetic manipulation.

The terms “treating” and “treatment” as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage. The terms “preventing” and “prevention” refer to the administration of an agent or composition to a clinically asymptomatic individual who is susceptible to a particular adverse condition, disorder, or disease, and thus relates to the prevention of the occurrence of symptoms and/or their underlying cause.

By the terms “effective amount” and “therapeutically effective amount” of a formulation or formulation component is meant a nontoxic but sufficient amount of the formulation or component to provide the desired effect.

The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

The host organism used to express the lactose-accepting fucosyltransferase gene is typically the enterobacterium Escherichia coli K12 (E. coli). E. coli K-12 is not considered a human or animal pathogen nor is it toxicogenic. E. coli K-12 is a standard production strain of bacteria and is noted for its safety due to its poor ability to colonize the colon and establish infections (see, e.g., epa.gov/oppt/biotech/pubs/fra/fra004.htm). However, a variety of bacterial species may be used in the oligosaccharide biosynthesis methods, e.g., Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentos, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus may be modified using the methods of this invention, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also included as part of this invention are strains, modified as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and a fucosylated oligosaccharide is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. The fucosylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacteria are used directly in such products. A suitable production host bacterial strain is one that is not the same bacterial strain as the source bacterial strain from which the fucosyltransferase-encoding nucleic acid sequence was identified.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing the synthetic pathway of the major neutral fucosyl-oligosaccharides found in human milk.

FIG. 2 is a schematic demonstrating metabolic pathways and the changes introduced into them to engineer 2′-fucosyllactose (2′-FL) synthesis in Escherichia coli (E. coli). Specifically, the lactose synthesis pathway and the GDP-fucose synthesis pathway are illustrated. In the GDP-fucose synthesis pathway: manA=phosphomannose isomerase (PMI), manB=phosphomannomutase (PMM), manC=mannose-1-phosphate guanylyltransferase (GMP), gmd=GDP-mannose-4,6-dehydratase, fcl=GDP-fucose synthase (GFS), and ΔwcaJ=mutated UDP-glucose lipid carrier transferase.

FIG. 3A and FIG. 3B show the sequence identity and a multiple sequence alignment of 4 previously known lactose-utilizing α(1,2)-fucosyltransferase protein sequences. FIG. 3A is a table showing the sequence identity between the 4 known lactose-utilizing α(1,2)-fucosyltransferases: H. pylori futC (SEQ ID NO: 1), H. mustelae FutL (SEQ ID NO: 2), Bacteroides vulgatus futN (SEQ ID NO: 3), and E. coli 0126 wbgL (SEQ ID NO: 4). FIG. 3B shows multiple sequence alignment of the 4 known α(1,2)-fucosyltransferases. The ovals highlight regions of particularly high sequence conservation between the four enzymes in the alignment.

FIG. 4 shows the sequence alignment of the 12 identified α(1,2)-fucosyltransferase syngenes identified, along with the 4 previously known lactose-utilizing α(1,2)-fucosyltransferase protein sequences. The 4 known lactose-utilizing α(1,2)-fucosyltransferases are boxed and include H. pylori futC (SEQ ID NO: 1), H. mustelae FutL (SEQ ID NO: 2), Bacteroides vulgatus futN (SEQ ID NO: 3), and E. coli 0126 wbgL (SEQ ID NO: 4). The 12 identified α(1,2)-fucosyltransferase are as follows: Prevotella melaninogenica FutO (SEQ ID NO: 10), Clostridium bolteae+13 FutP (SEQ ID NO: 292), Lachnospiraceae sp. FutQ (SEQ ID NO: 12), Methanosphaerula palustris FutR (SEQ ID NO: 13), Tannerella sp. FutS (SEQ ID NO: 14), Bacteroides caccae FutU (SEQ ID NO: 15), Butyrivibrio FutV (SEQ ID NO: 16), Prevotella sp. FutW (SEQ ID NO: 17), Parabacteroides johnsonii FutX (SEQ ID NO: 18), Akkermansia muciniphilia FutY (SEQ ID NO: 19), Salmonella enterica FutZ (SEQ ID NO: 20), Bacteroides sp. FutZA (SEQ ID NO: 21). The sequence for Clostridium bolteae FutP (without the 13 additional amino acids in the N-terminus) (SEQ ID NO: 11) is also shown in the alignment.

FIG. 5A and FIG. 5B are two pictures of gels showing the construction of the syngenes for each of the 12 novel α(1,2)-fucosyltransferases. FIG. 5A shows post-Gibson assembly PCR. FIG. 5B shows gel-purified RI/Xho 1 syngene fragments.

FIG. 6A and FIG. 6B are two photographs showing thin layer chromatograms of fucosylated oligosaccharide products produced in E. coli cultures using the 12 novel α(1,2)-fucosyltransferase syngenes. FIG. 6A shows fucosylated oligosaccharide products from 2 μl of culture supernatant. FIG. 6B shows fucosylated oligosaccharide products from 0.2 OD₆₀₀ cell equivalents of whole cell heat extracts.

FIG. 7 is a graph showing the growth curve of the host bacterium expressing plasmids containing the α(1,2) fucosyltransferase genes WbgL, FutN, FutO, FutQ, and FutX after tryptophan induction in the presence of lactose in the culture medium (i.e. lac+trp).

FIG. 8 is a photograph of a SDS-PAGE gel showing the proteins produced from host bacterium expressing α(1,2) fucosyltransferase genes WbgL, FutN, FutO, FutQ, and FutX after induction.

FIG. 9A and FIG. 9B are two photographs of thin layer chromatograms showing the production of fucosylated oligosaccharide products from in E. coli cultures expressing select α(1,2)-fucosyltransferase syngenes WbgL, FutN, FutO, FutQ, and FutX at 7 hours or 24 hours after induction. FIG. 9A shows fucosylated oligosaccharide products from 2 μl of culture supernatant. FIG. 9B shows fucosylated oligosaccharide products from 0.2 OD₆₀₀ cell equivalents of whole cell heat extracts.

FIG. 10A and FIG. 10B are two photographs of thin layer chromatograms showing the fucosylated oligosaccharide products after two different 1.5 L fermentation runs from E. coli expressing FutN: FIG. 10A) 36B and FIG. 10B) 37A. The culture yield for run 36B was 33 g/L while the yield for run 37A was 36.3 g/L.

FIG. 11 is a plasmid map of pG217 carrying the B. vulgatus FutN gene.

FIG. 12 is a schematic diagram showing the insertion of the LacIq promoter, the functional lacY gene, and the deletion of lacA.

FIG. 13 is a schematic diagram showing the deletion of the endogenous wcaJ gene using FRT recombination.

FIG. 14 is a schematic diagram of the E. coli W3110 chromosome, showing the insertion of a DNA fragment carrying kanamycin resistance gene (derived from transposon Tn5) and wild-type lacZ into the lon gene.

DETAILED DESCRIPTION OF THE INVENTION

While some studies suggest that human milk glycans could be used as antimicrobial anti-adhesion agents, the difficulty and expense of producing adequate quantities of these agents of a quality suitable for human consumption has limited their full-scale testing and perceived utility. What has been needed is a suitable method for producing the appropriate glycans in sufficient quantities at reasonable cost. Prior to the invention described herein, there were attempts to use several distinct synthetic approaches for glycan synthesis. Some chemical approaches can synthesize oligosaccharides (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003)), but reactants for these methods are expensive and potentially toxic (Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). Enzymes expressed from engineered organisms (Albermann, C., Piepersberg, W. & Wehmeier, U. F. Carbohydr Res 334, 97-103 (2001); Bettler, E., Samain, E., Chazalet, V., Bosso, C., et al. Glycoconj J 16, 205-212 (1999); Johnson, K. F. Glycoconj J 16, 141-146 (1999); Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999); Wymer, N. & Toone, E. J. Curr Opin Chem Biol 4, 110-119 (2000)) provide a precise and efficient synthesis (Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999)); Crout, D. H. & Vic, G. Curr Opin Chem Biol 2, 98-111 (1998)), but the high cost of the reactants, especially the sugar nucleotides, limits their utility for low-cost, large-scale production. Microbes have been genetically engineered to express the glycosyltransferases needed to synthesize oligosaccharides from the bacteria's innate pool of nucleotide sugars (Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 330, 439-443 (2001); Endo, T., Koizumi, S., Tabata, K. & Ozaki, A. Appl Microbiol Biotechnol 53, 257-261 (2000); Endo, T. & Koizumi, S. Curr Opin Struct Biol 10, 536-541 (2000); Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 316, 179-183 (1999); Koizumi, S., Endo, T., Tabata, K. & Ozaki, A. Nat Biotechnol 16, 847-850 (1998)). However, prior to the invention described herein, there was a growing need to identify and characterize additional glycosyltransferases that are useful for the synthesis of HMOS in metabolically engineered bacterial hosts.

Human Milk Glycans

Human milk contains a diverse and abundant set of neutral and acidic oligosaccharides (Kunz, C., Rudloff, S., Baier, W., Klein, N., and Strobel, S. (2000). Annu Rev Nutr 20, 699-722; Bode, L. (2006). J Nutr 136, 2127-130). More than 130 different complex oligosaccharides have been identified in human milk, and their structural diversity and abundance is unique to humans. Although these molecules may not be utilized directly by infants for nutrition, they nevertheless serve critical roles in the establishment of a healthy gut microbiome (Marcobal, A., Barboza, M., Froehlich, J. W., Block, D. E., et al. J Agric Food Chem 58, 5334-5340 (2010)), in the prevention of disease (Newburg, D. S., Ruiz-Palacios, G. M. & Morrow, A. L. Annu Rev Nutr 25, 37-58 (2005)), and in immune function (Newburg, D. S. & Walker, W. A. Pediatr Res 61, 2-8 (2007)). Despite millions of years of exposure to human milk oligosaccharides (HMOS), pathogens have yet to develop ways to circumvent the ability of HMOS to prevent adhesion to target cells and to inhibit infection. The ability to utilize HMOS as pathogen adherence inhibitors promises to address the current crisis of burgeoning antibiotic resistance. Human milk oligosaccharides produced by biosynthesis represent the lead compounds of a novel class of therapeutics against some of the most intractable scourges of society.

One alternative strategy for efficient, industrial-scale synthesis of HMOS is the metabolic engineering of bacteria. This approach involves the construction of microbial strains overexpressing heterologous glycosyltransferases, membrane transporters for the import of precursor sugars into the bacterial cytosol, and possessing enhanced pools of regenerating nucleotide sugars for use as biosynthetic precursors (Dumon, C., Samain, E., and Priem, B. (2004). Biotechnol Prog 20, 412-19; Ruffing, A., and Chen, R. R. (2006). Microb Cell Fact 5, 25). A key aspect of this approach is the heterologous glycosyltransferase selected for overexpression in the microbial host. The choice of glycosyltransferase can significantly affect the final yield of the desired synthesized oligosaccharide, given that enzymes can vary greatly in terms of kinetics, substrate specificity, affinity for donor and acceptor molecules, stability and solubility. A few glycosyltransferases derived from different bacterial species have been identified and characterized in terms of their ability to catalyze the biosynthesis of HMOS in E. coli host strains (Dumon, C., Bosso, C., Utille, J. P., Heyraud, A., and Samain, E. (2006). Chembiochem 7, 359-365; Dumon, C., Samain, E., and Priem, B. (2004). Biotechnol Prog 20, 412-19; Li, M., Liu, X. W., Shao, J., Shen, J., Jia, Q., Yi, W., Song, J. K., Woodward, R., Chow, C. S., and Wang, P. G. (2008). Biochemistry 47, 378-387). The identification of additional glycosyltransferases with faster kinetics, greater affinity for nucleotide sugar donors and/or acceptor molecules, or greater stability within the bacterial host significantly improves the yields of therapeutically useful HMOS. Prior to the invention described herein, chemical syntheses of HMOS were possible, but were limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003); Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). The invention overcomes the shortcomings of these previous attempts by providing new strategies to inexpensively manufacture large quantities of human milk oligosaccharides (HMOS) for use as dietary supplements. Advantages include efficient expression of the enzyme, improved stability and/or solubility of the fucosylated oligosaccharide product (2′-FL, LDFT, LNF I, and LDFH I) and reduced toxicity to the host organism. The present invention features novel α(1,2) FTs suitable for expression in production strains for increased efficacy and yield of fucosylated HMOS compared to α(1,2) FTs currently utilized in the field.

As described in detail below, E. coli (or other bacteria) is engineered to produce selected fucosylated oligosaccharides (i.e., 2′-FL, LDFT, LDHF I, or LNF I) in commercially viable levels. For example, yields are >5 grams/liter in a bacterial fermentation process. In other embodiments, the yields are greater than 10 grams/liter, greater than 15 grams/liter, greater than 20 grams/liter, greater than 25 grams/liter, greater than 30 grams/liter, greater than 35 grams/liter, greater than 40 grams/liter, greater than 45 grams/liter, greater than 50 grams/liter, greater than 55 grams/liter, greater than 60 grams/liter, greater than 65 grams/liter, greater than 70 grams/liter, or greater than 75 grams/liter of fucosylated oligosaccharide products, such as 2′-FL, LDFT, LDHF I, and LNF I.

Role of Human Milk Glycans in Infectious Disease

Human milk glycans, which comprise both unbound oligosaccharides and their glycoconjugates, play a significant role in the protection and development of the infant gastrointestinal (GI) tract. Neutral fucosylated oligosaccharides, including 2′-fucosyllactose (2′-FL), protect infants against several important pathogens. Milk oligosaccharides found in various mammals differ greatly, and the composition in humans is unique (Hamosh M., 2001 Pediatr Clin North Am, 48:69-86; Newburg D. S., 2001 Adv Exp Med Biol, 501:3-10). Moreover, glycan levels in human milk change throughout lactation and also vary widely among individuals (Morrow A. L. et al., 2004 J Pediatr, 145:297-303; Chaturvedi P et al., 2001 Glycobiology, 11:365-372). Approximately 200 distinct human milk oligosaccharides have been identified and combinations of simple epitopes are responsible for this diversity (Newburg D. S., 1999 Curr_Med Chem, 6:117-127; Ninonuevo M. et al., 2006 J Agric Food Chem, 54:7471-74801).

Human milk oligosaccharides are composed of 5 monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetyl neuraminic acid, Neu5Ac, NANA). Human milk oligosaccharides are usually divided into two groups according to their chemical structures: neutral compounds containing Glc, Gal, GlcNAc, and Fuc, linked to a lactose (Galβ1-4Glc) core, and acidic compounds including the same sugars, and often the same core structures, plus NANA (Charlwood J. et al., 1999 Anal Biochem, 273:261-277; Martín-Sosa et al., 2003 J Dairy Sci, 86:52-59; Parkkinen J. and Finne J., 1987 Methods Enzymol, 138:289-300; Shen Z. et al., 2001 J Chromatogr A, 921:315-321).

Approximately 70-80% of oligosaccharides in human milk are fucosylated, and their synthetic pathways are believed to proceed as shown in FIG. 1. A smaller proportion of the oligosaccharides are sialylated or both fucosylated and sialylated, but their synthetic pathways are not fully defined. Understanding of the acidic (sialylated) oligosaccharides is limited in part by the ability to measure these compounds. Sensitive and reproducible methods for the analysis of both neutral and acidic oligosaccharides have been designed. Human milk oligosaccharides as a class survive transit through the intestine of infants very efficiently, being essentially indigestible (Chaturvedi, P., Warren, C. D., Buescher, C. R., Pickering, L. K. & Newburg, D. S. Adv Exp Med Biol 501, 315-323 (2001)).

Human Milk Glycans Inhibit Binding of Enteropathogens to their Receptors

Human milk glycans have structural homology to cell receptors for enteropathogens and function as receptor decoys. For example, pathogenic strains of Campylobacter bind specifically to glycans containing H-2, i.e., 2′-fucosyl-N-acetyllactosamine or 2′-fucosyllactose (2′FL); Campylobacter binding and infectivity are inhibited by 2′-FL and other glycans containing this H-2 epitope. Similarly, some diarrheagenic E. coli pathogens are strongly inhibited in vivo by human milk oligosaccharides containing 2-linked fucose moieties. Several major strains of human caliciviruses, especially the noroviruses, also bind to 2-linked fucosylated glycans, and this binding is inhibited by human milk 2-linked fucosylated glycans. Consumption of human milk that has high levels of these 2-linked fucosyloligosaccharides was associated with lower risk of norovirus, Campylobacter, ST of E. coli-associated diarrhea, and moderate-to-severe diarrhea of all causes in a Mexican cohort of breastfeeding children (Newburg D. S. et al., 2004 Glycobiology, 14:253-263; Newburg D. S. et al., 1998 Lancet, 351:1160-1164). Several pathogens utilize sialylated glycans as their host receptors, such as influenza (Couceiro, J. N., Paulson, J. C. & Baum, L. G. Virus Res 29, 155-165 (1993)), parainfluenza (Amonsen, M., Smith, D. F., Cummings, R. D. & Air, G. M. J Virol 81, 8341-8345 (2007), and rotoviruses (Kuhlenschmidt, T. B., Hanafin, W. P., Gelberg, H. B. & Kuhlenschmidt, M. S. Adv Exp Med Biol 473, 309-317 (1999)). The sialyl-Lewis X epitope is used by Helicobacter pylori (Mandavi, J., Sondén, B., Hurtig, M., Olfat, F. O., et al. Science 297, 573-578 (2002)), Pseudomonas aeruginosa (Scharfman, A., Delmotte, P., Beau, J., Lamblin, G., et al. Glycoconj J 17, 735-740 (2000)), and some strains of noroviruses (Rydell, G. E., Nilsson, J., Rodriguez-Diaz, J., Ruvoën-Clouet, N., et al. Glycobiology 19, 309-320 (2009)).

Identification of Novel α(1,2) Fucosyltransferases

The present invention provides novel α(1,2) fucosyltransferase enzymes. The present invention also provides nucleic acid constructs (i.e., a plasmid or vector) carrying the nucleic acid sequence of a novel α(1,2) fucosyltransferases for the expression of the novel α(1,2) fucosyltransferases in host bacterium. The present invention also provides methods for producing fucosylated oligosaccharides by expressing the novel α(1,2) fucosyltransferases in suitable host production bacterium, as further described herein.

Not all α(1,2)fucosyltransferases can utilize lactose as an acceptor substrate. An acceptor substrate includes, for example, a carbohydrate, an oligosaccharide, a protein or glycoprotein, a lipid or glycolipid, e.g., N-acetylglucosamine, N-acetyllactosamine, galactose, fucose, sialic acid, glucose, lactose, or any combination thereof. A preferred alpha (1,2) fucosyltransferase of the present invention utilizes GDP-fucose as a donor, and lactose is the acceptor for that donor.

A method of identifying novel α(1,2)fucosyltransferase enzymes capable of utilizing lactose as an acceptor was previously carried out (as described in PCT/US2013/051777, hereby incorporated by reference in its entirety) using the following steps: 1) performing a computational search of sequence databases to define a broad group of simple sequence homologs of any known, lactose-utilizing α(1,2)fucosyltransferase; 2) using the list of homologs from step 1 to derive a search profile containing common sequence and/or structural motifs shared by the members of the broad group, e.g. by using computer programs such as MEME (Multiple Em for Motif Elicitation available at http://meme.sdsc.edu/meme/cgi-bin/meme.cgi) or PSI-BLAST (Position-Specific Iterated BLAST available at ncbi.nlm.nih.gov/blast with additional information at cnx.org/content/m11040/latest/); 3) searching sequence databases (e.g., using computer programs such as PSI-BLAST, or MAST (Motif Alignment Search Tool available at http://meme.sdsc.edu/meme/cgi-bin/mast.cgi); using this derived search profile as query, and identifying “candidate sequences” whose simple sequence homology to the original lactose-accepting α(1,2)fucosyltransferase is 40% or less; 4) scanning the scientific literature and developing a list of “candidate organisms” known to express α(1,2)fucosyl-glycans; 5) selecting only those “candidate sequences” that are derived from “candidate organisms” to generate a list of “candidate lactose-utilizing enzymes”; and 6) expressing each “candidate lactose-utilizing enzyme” and testing for lactose-utilizing α(1,2)fucosyltransferase activity.

The MEME suite of sequence analysis tools (meme.sdsc.edu/meme/cgi-bin/meme.cgi) can also be used as an alternative to PSI-BLAST. Sequence motifs are discovered using the program “MEME”. These motifs can then be used to search sequence databases using the program “MAST”. The BLAST and PSI-BLAST search algorithms are other well known alternatives.

To identify additional novel α(1,2)fucosyltransferases, a multiple sequence alignment query was generated using four previously identified lactose-utilizing α(1,2)fucosyltransferase protein sequences: H. pylori futC (SEQ ID NO: 1), H. mustelae FutL (SEQ ID NO: 2), Bacteroides vulgatus futN (SEQ ID NO: 3), and E. coli 0126 wbgL (SEQ ID NO: 4). These sequence alignment and percentage of sequence identity is shown in FIG. 3. An iterative PSI-BLAST was performed, using the FASTA-formatted multiple sequence alignment as the query, and the NCBI PSI-BLAST program run on a local copy of NCBI BLAST+ version 2.2.29. An initial position-specific scoring matrix file (.pssm) was generated by PSI-BLAST, which the program then used to adjust the score of iterative homology search runs. The process is iterated to generate an even larger group of candidates, and the results of each run were used to further refine the matrix.

This PSI-BLAST search resulted in an initial 2515 hits. There were 787 hits with greater than 22% sequence identity to FutC. 396 hits were of greater than 275 amino acids in length. Additional analysis of the hits was performed, including sorting by percentage identity to FutC, comparing the sequences by BLAST to existing α(1,2) fucosyltransferase inventory (of known α(1,2) fucosyltransferases), and manual annotation of hit sequences to identify those originating from bacteria that naturally exist in the gastrointestinal tract. An annotated list of the novel α(1,2) fucosyltransferases identified by this screen are listed in Table 1. Table 1 provides the bacterial species from which the candidate enzyme is found, the GenBank Accession Number, GI Identification Number, amino acid sequence, and % sequence identity to FutC.

Of the identified hits, 12 novel α(1,2) fucosyltransferases were further analyzed for their functional capacity: Prevotella melaninogenica FutO, Clostridium bolteae FutP, Clostridium bolteae+13 FutP, Lachnospiraceae sp. FutQ, Methanosphaerula palustries FutR, Tannerella sp. FutS, Bacteroides caccae FutU, Butyrivibrio FutV, Prevotellaa sp. FutW, Parabacteroides johnsonii FutX, Akkermansia muciniphilia FutY, Salmonella enterica FutZ, and Bacteroides sp. FutZA. For Clostridium bolteae FutP, the annotation named the wrong initiation methionine codon. Thus, the present invention includes FutP with an additional 13 amino acids at the N-terminus of the annotated FutP (derived in-frame from the natural upstream DNA sequence), which is designated herein as Clostridium bolteae+13 FutP. The sequence identity between the 12 novel α(1,2) fucosyltransferases identified and the 4 previously identified α(1,2) fucosyltransferases is shown in Table 2 below.

TABLE 2 Sequence Identity 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 H. pylori futC 1 70.10 21.99 20.82 27.68 27.36 23.56 23.26 23.62 25.75 23.72 24.05 12.29 24.19 22.92 22.29 H. mustelae futL 2 70.10 23.87 19.88 26.38 28.21 24.30 23.38 24.62 25.31 25.31 24.47 23.56 25.15 23.55 23.26 Bacteroides vulgatus futN 3 21.99 23.87 25.16 32.05 28.71 28.94 25.79 37.46 32.27 26.11 61.27 71.63 27.67 25.15 84.75 E. coli O126 wbgL 4 20.82 19.88 25.16 24.25 22.73 22.32 26.04 25.45 24.77 21.49 73.29 26.71 24.63 21.45 25.16 Prevotella melaninogenica 5 27.68 26.36 32.05 24.25 36.96 31.63 35.74 35.16 55.74 30.28 30.03 32.80 30.09 26.28 31.83 FutO YP_003814512.1 Clostridium bolteae + 6 27.36 28.21 28.71 22.73 36.96 37.87 35.10 33.77 36.91 35.74 29.53 31.39 27.67 26.33 29.13 13 FutP WP_002570768.1 Lachnospiraceae sp. 7 23.56 24.30 28.94 22.32 31.63 37.87 29.87 29.17 32.90 51.02 28.53 30.00 27.69 24.00 27.74 FutQ WP_009251343.1 Methanospharula palustris 8 23.28 23.38 25.79 26.04 35.74 35.10 29.87 18.71 38.24 31.41 25.39 28.08 30.65 23.93 25.55 FutR YP_002467213.1 Tannerella sp. 9 23.62 24.62 37.46 25.45 35.16 33.77 29.17 28.71 34.41 30.03 35.71 36.27 26.48 21.75 36.60 FutS WP_021929367.1 Bacteroides caccae 10 25.75 25.31 32.27 24.77 55.74 36.91 32.90 38.24 34.41 31.21 29.94 33.33 29.28 24.40 33.01 FutU WP_005675707.1 Butyrivibrio 11 23.72 25.31 25.11 21.49 30.28 35.74 51.02 31.41 30.03 31.21 27.62 26.20 26.46 22.15 26.52 FutV WP_022772718.1 Prevotella sp. 12 24.05 24.47 61.27 23.29 30.03 29.58 28.53 25.39 15.71 29.94 27.62 57.60 25.79 22.15 59.01 FutV WP_022481266.1 Parabacteroides johnsonni 13 22.29 23.56 71.63 26.71 32.80 31.39 30.00 28.08 36.27 33.33 26.20 57.60 28.71 24.00 74.02 FutX WP_008155883.1 Akkermansia muciniphilia 14 24.19 25.15 27.67 24.63 30.09 27.67 27.69 30.65 26.41 29.28 26.46 25.79 28.71 21.45 28.08 FutY YP_00187755.5 Salmonella enterica 15 21.92 23.55 25.15 21.45 26.24 26.33 14.00 23.93 21.75 24.46 22.13 22.15 24.00 21.45 74.62 FutZ WP_023214330 Bacteroides sp. 16 22.29 23.26 84.75 25.16 31.83 29.13 27.74 25.55 35.50 33.01 26.52 59.01 74.02 28.08 24.62 FutZA WP_022161880.11

Based on the amino acid sequences of the identified α(1,2) fucosyltransferases (i.e., in Table 1), syngenes can be readily designed and constructed by the skilled artisan using standard methods known in the art. For example, the syngenes include a ribosomal binding site, are codon-optimized for expression in a host bacterial production strain (i.e., E. coli), and have common 6-cutter restriction sites or sites recognized by endogenous restriction enzymes present in the host strain (i.e., EcoK restriction sites) removed to ease cloning and expression in the E. coli host strain. In a preferred embodiment, the syngenes are constructed with the following configuration: EcoRI site-T7g10 RBS-α(1,2) FT syngene-XhoI site. The nucleic acid sequences of sample syngenes for the 12 identified α(1,2) fucosyltransferases are shown in Table 3. (the initiating methionine ATG codon is bolded)

TABLE 3 Nucleic acid sequences of 12 novel a(1,2) fucosyltransferase syngenes Bacteria/ SEQ Gene ID name Sequence NO: FutO CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGAAAATCGTCAAAATCCTGGGCGGT 276 CTGGGCAATCAGATGTTCCAGTATGCTCTGTACCTGAGCCTGCAAGAAAGTTTTCCAAAA GAACGTGTGGCCCTGGACCTGTCCTCCTTCCACGGCTATCACCTGCATAATGGCTTTGAG CTGGAGAACATTTTCTCCGTTACCGCTCAGAAAGCATCCGCCGCAGATATCATGCGTATT GCTTATTACTACCCGAACTATCTGCTGTGGCGCATTGGCAAACGTTTTCTGCCGCGTCGT AAAGGTATGTGCCTGGAATCTAGCTCCCTGCGTTTCGATGAAAGCGTTCTGCGTCAGGAA GGTAACCGTTATTTTGACGGTTACTGGCAAGACGAACGCTACTTCGCAGCCTATCGTGAA AAAGTGCTGAAGGCTTTCACCTTTCCTGCATTCAAACGCGCAGAAAACCTGAGCCTGCTG GAAAAACTGGACGAAAACAGCATTGCTCTGCATGTTCGTCGCGGTGATTACGTAGGTAAT AACCTGTACCAAGGCATCTGTGACCTGGACTACTACCGTACCGCTATCGAGAAAATGTGT GCACACGTTACTCCGTCTCTGTTTTGTATCTTTTCCAACGACATCACGTGGTGCCAGCAG CACCTGCAACCGTACCTGAAGGCCCCTGTGGTGTACGTTACTTGGAACACCGGTGTTGAA TCCTACCGCGATATGCAGCTGATGTCCTGCTGCGCACATAACATCATCGCGAATAGCTCC TTCTCTTGGTGGGGTGCTTGGCTGAATCAGAACCGTGAAAAAGTTGTTATCGCCCCGAAA AAATGGCTGAACATGGAAGAATGTCACTTCACGCTGCCGGCAAGCTGGATCAAAATTTAG CTCGAGTGACTGACTG FutP CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGGTGATTATCAAAATGATGGGTGGT 277 CTGGGCAACCAGATGTTCCAGTACGCACTGTACAAAGCATTCGAGCAGAAGCACATCGAT GTGTATGCAGACCTGGCATGGTACAAAAACAAATCCGTGAAATTTGAACTGTACAACTTC GGCATTAAAATCAACGTAGCATCCGAGAAAGACATCAACCGTCTGAGCGATTGCCAGGCG GACTTTGTTTCCCGCATCCGCCGTAAAATCTTTGGTAAAAAAAAGAGCTTCGTATCTGAA AAAAATGACTCCTGCTATGAAAACGACATCCTGCGTATGGACAACGTTTATCTGAGCGGT TATTGGCAGACCGAAAAATACTTCTCTAACACGCGTGAGAAGCTGCTGGAGGATTATTCC TTCGCTCTGGTAAACTCTCAGGTGTCCGAATGGGAAGACTCCATTCGCAACAAAAACAGC GTTAGCATCCATATCCGTCGTGGTGATTATCTACAGGGCGAACTGTATGGTGGTATTTGC ACCTCTCTGTACTACGCCGAAGCAATCGAGTACATTAAAATGCGTGTTCCGAACGCAAAA TTCTTCGTTTTCTCTGATGACGTTGAATGGGTTAAACAGCAAGAAGACTTCAAAGGCTTC GTAATCGTTGATCGCAACGAGTATTCTAGCGCTCTGTCCGATATGTACCTGATGTCCCTG TGCAAGCATAACATTATTGCTAACTCCTCTTTCAGCTGGTGGGCAGCTTGGCTGAACCGT AACGAAGAAAAAATTGTAATCGCGCCGCGCCGTTGGCTGAACGGCAAGTGCACCCCAGAT ATCTGGTGTAAAAAATGGATTCGTATCTAGCTCGAGTGACTGACTG FutQ CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGGTGATCGTACAGCTGAGCGGCGGT 278 CTGGGCAACCAGATGTTCGAATACGCGCTGTACCTGAGCCTGAAAGCAAAAGGCAAAGAA GTGAAAATTGACGATGTTACGTGTTACGAGGGCCCTGGCACCCGTCCGCGTCAACTGGAT GTTTTTGGTATCACGTACGATCGCGCGTCTCGTGAGGAGCTGACTGAGATGACGGACGCG AGCATGGATGCGCTGTCTCGTGTTCGTCGCAAACTGACCGGTCGCCGCACTAAAGCGTAC CGCGAACGCGACATCAACTTCGATCCACTGGTTATGGAAAAAGACCCGGCACTGCTGGAA GGCTGTTTCCAGTCTGACAAATACTTTCGTGATTGCGAAGGCCGCGTGCGCGAACCGTAT CGTTTCCGCGGCATTGAATCCGGCGCGTTCCCGCTGCCGGAAGACTATCTGCGCCTGGAA AAGCAGATCGAAGATTGTCAGTCCGTATCCGTACACATCCGTCGTGGCGACTACCTGGAC GAATCTCATGGTGGTCTGTACACCGGCATTTGTACTGAGGCGTACTATAAAGAGGCTTTT GCTCGCATGGAACGTCTGGTTCCGGGCGCACGTTTCTTCCTGTTCTCTAACGATCCAGAA TGGACTCGTGAGCACTTTGAGAGCAAGAACTGCGTTCTGGTTGAAGGTAGCACCGAAGAC ACGGGTTACATGGACCTGTACCTGATGAGCCGCTGCCGCCACAATATTATTGCCAACTCT TCTTTCAGCTGGTGGGGCGCTTGGCTGAATGAGAACCCTGAGAAAAAAGTCATCGCACCG GCTAAATGGCTGAACGGTCGTGAGTGCCGTGATATCTATACCGAACGCATGATTCGTCTG TAGCTCGAGTGACTGACTG FutR CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGATCATTGTTCGTCTGAAAGGCGGT 279 CTGGGCAACCAACTGTCTCAGTATGCACTGGGCCGTAAGATCGCGCATCTGCACAATACC GAACTGAAACTGGACACCACTTGGTTCACCACTATCTCCTCCGACACTCCACGTACCTAC CGTCTGAACAATTATAACATCATCGGCACTATTGCATCCGCAAAGGAAATCCAGCTGATC GAACGTGGTCGCGCGCAAGGCCGTGGCTACCTGCTGTCTAAAATTTCTGATCTGCTGACT CCGATGTACCGTCGTACCTACGTCCGTGAACGTATGCATACCTTCGATAAAGCTATCCTG ACCGTTCCGGACAACGTGTACCTGGATGGTTACTGGCAGACCGAAAAGTACTTCAAAGAC ATCGAAGAAATCCTGCGCCGTGAGGTTACGCTGAAAGATGAACCGGATAGCATCAACCTG GAAATGGCTGAACGTATTCAGGCTTGCCACAGCGTTTCCCTGCACGTGCGTCGTGGCGAC TACGTTTCCAACCCGACCACTCAACAATTCCACGGCTGTTGCTCCATTGACTACTACAAC CGCGCTATCTCTCTGATTGAAGAAAAAGTGGATGACCCGTCTTTCTTTATTTTTTCTGAC GATCTGCCGTGGGCTAAAGAAAACCTGGACATCCCTGGCGAAAAAACCTTCGTTGCGCAT AACGGCCCGGAAAAAGAGTATTGCGATCTGTGGCTGATGTCTCTGTGCCAGCACCATATC ATCGCAAACTCTTCTTTCAGCTGGTGGGGTGCCTGGCTGGGTCAAGACGCCGAAAAGATG GTGATCGCGCCGCGTCGCTGGGCCCTGTCCGAGAGCTTTGACACTTCTGACATCATTCCG GACTCTTGGATTACTATCTAGCTCGAGTGACTGACTG FutS CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGGTACGCATTGTGGAAATCATCGGC 280 GGTCTGGGTAACCAGATGTTCCAGTACGCATTCTCCCTGTACCTGAAAAACAAATCTCAC ATCTGGGACCGTCTGTATGTGGACATCGAGGCGATGAAAACCTACGATCGTCACTATGGT CTGGAACTGGAGAAAGTTTTCAATCTGAGCCTGTGTCCAATCTCTAACCGTCTGCACCGC AACCTGCAAAAACGCTCCTTCGCAAAACACTTTGTAAAGAGCCTGTACGAGCACTCTGAA TGCGAGTTCGACGAACCGGTGTACCGTGGCCTGCGTCCTTATCGCTATTATCGCGGCTAC TGGCAAAACGAAGGTTACTTCGTTGATATTGAACCGATGATCCGTGAGGCTTTTCAGTTC AACGTTAACATCCTGAGCAAAAAGACTAAAGCGATCGCATCCAAAATGCGCCGTGAACTG TCCGTATCTATCCATGTTCGCCGTGGTGATTACGAAAACCTGCCGGAAGCGAAAGCGATG CATGGCGGTATTTGTTCTCTGGACTATTACCACAAAGCGATCGACTTCATCCGCCAGCGT CTGGATAATAACATCTGTTTCTATCTGTTCTCCGACGATATCAATTGGGTAGAAGAAAAC CTGCAACTGGAAAACCGTTGCATCATCGACTGGAACCAGGGCGAAGATAGCTGGCAGGAC ATGTACCTGATGAGCTGCTGCCGCCACCACATTATCGCAAACAGCTCTTTCTCCTGGTGG GCGGCATGGCTGAATCCAAACAAGAACAAAATCGTACTGACCCCGAACAAATGGTTCAAC CATACTGACGCAGTGGGTATCGTCCCAAAGTCCTGGATTAAAATTCCTGTGTTTTAGCTC GAGTGACTGACTG FutU CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGAAAATCGTTAAAATCCTGGGCGGC 281 CTGGGTAACCAGATGTTTCAGTACGCCCTGTTCCTGTCTCTGAAAGAACGCTTCCCGCAT GAACAGGTGATGATTGACACCAGCTGCTTCCGCAATTACCCACTGCACAACGGTTTCGAA GTGGATCGTATCTTCGCCCAGAAAGCACCGGTTGCCTCTTGGCGTAACATCCTGAAGGTT GCCTACCCGTACCCGAACTACCGTTTCTGGAAAATCGGTAAATACATCCTGCCTAAACGT AAAACCATGTGTGTAGAGCGTAAAAACTTCAGCTTTGACGCCGCAGTCCTGACCCGTAAA GGCGATTGCTACTATGATGGCTACTGGCAGCATGAGGAATATTTCTGTGATATGAAAGAA ACGATTTGGGAGGCTTTCTCCTTCCCTGAGCCGGTTGATGGTCGTAACAAGGAGATCGGT GCCCTGCTACAGGCATCTGATAGCGCTTCCCTGCACGTTCGTCGCGGTGACTACGTGAAC CACCCACTGTTTCGTGGTATTTGTGACCTGGACTATTATAAACGTGCCATCCACTACATG GAAGAACGCGTCAACCCACAGCTGTACTGCGTTTTCAGCAACGATATGGCCTGGTGCGAG TCCCACCTGCGTGCACTGCTGCCAGGCAAAGAAGTAGTTTATGTTGACTGGAACAAGGGT GCGGAATCTTACGTTGATATGCGTCTGATGAGCCTGTGCCGTCACAACATCATCGCTAAC TCTTCTTTCAGCTGGTGGGGCGCATGGCTGAACCGTAACCCGCAGAAAGTGGTGGTAGCG CCGGAACGTTGGATGAACAGCCCGATTGAAGACCCAGTGAGCGACAAATGGATTAAACTG TAGCTCGAGTGACTGACTG FutV CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGATCATCATCCAGCTGAAAGGTGGC 282 CTGGGCAACCAAATGTTCCAGTACGCGCTGTACAAATCCCTGAAAAAACGTGGTAAAGAA GTTAAAATTGATGACAAAACTGGCTTCGTGAACGACAAACTGCGTATCCCGGTACTGTCC CGTTGGGGTGTTGAGTACGATCGTGCAACCGACGAAGAGATTATTAACCTGACCGACTCC AAAATGGACCTGTTCTCTCGCATCCGCCGTAAACTGACTGGCCGCAAAACGTTCCGTATC GACGAAGAATCCGGTAAATTCAACCCGGAAATCCTGGAAAAAGAGAACGCTTATCTGGTG GGTTATTGGCAGTGCGACAAGTACTTCGACGACAAAGATGTGGTTCGCGAAATTCGTGAA GCGTTCGAGAAAAAACCGCAGGAGCTGATGACCGACGCCAGCTCTTGGTCTACTCTACAG CAGATTGAATGCTGCGAGTCCGTATCCCTGCACGTACGTCGTACTGATTACGTGGACGAG GAACATATTCATATCCATAACATCTGTACGGAAAAATACTATAAAAACGCCATTGATCGT GTGCGTAAACAGTACCCGAGCGCAGTGTTCTTCATCTTCACCGATGATAAAGAATGGTGC CGCGACCACTTTAAAGGTCCGAACTTCATCGTAGTCGAACTGGAAGAAGGCGACGGTACC GACATCGCTGAAATGACTCTGATGTCCCGCTGTAAACATCACATCATCGCTAATTCTAGC TTTAGCTGGTGGGCGGCGTGGCTGAACGACTCCCCGGAPAAAATCGTGATCGCTCCTCAG AAATGGATTAACAACCGCGACATGGACGATATTTACACCGAGCGTATGACTAAAATCGCA CTGTAGCTCGAGTGACTGACTG FutW CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGCGCCTGGTTAAAATGATCGGCGGT 283 CTGGGTAATCAGATGTTCATCTACGCGTTTTACCTACAGATGCGTAAGCGTTTCTCCAAC GTTCGTATCGACCTGACCGATATGATGCACTACAACGTACACTATGGCTACGAACTGCAC AAAGTTTTCGGTCTGCCGCGCACCGAGTTCTGTATGAACCAGCCTCTGAAAAAGGTTCTG GAGTTCCTGTTCTTCCGTACCATTGTTGAACGTAAACAGCACGGTCGTATGGAGCCGTAT ACTTGCCAGTATGTTTGGCCGCTGGTTTACTTTAAGGGCTTCTATCAGTCCGAACGTTAC TTCTCCGAAGTTAAGGACGAAGTTCGTGAGTGTTTCACCTTCAATCCGGCACTGGCGAAT CGTTCTTCCCAACAGATGATGGAACAGATCCAGAATGATCCTCAGGCTGTCTCTATCCAC ATCCGTCGTGGCGACTATCTGAATCCGAAGCACTACGACACTATCGGTTGTATCTGTCAG CTGCCGTATTACAAGCACGCCGTTTCCGAAATTAAAAAGTACGTTTCTAACCCTCACTTT TACGTTTTCTCCGAAGACCTGGATTGGGTCAAAGCAAACCTGCCGCTGGAAAACGCACAG TACATCGATTGGAACAAAGGCGCAGATAGCTGGCAGGATATGATGCTGATGAGCTGTTGC AAACACCACATTATCTGTAACTCCACCTTTAGCTGGTGGGCGGCGTGGCTGAACCCATCT GTCGAAAAAACCGTGATCATGCCGGAACAGTGGACGTCTCGTCAAGATTCCGTGGACTTT GTGGCTAGCTGTGGCCGTTGGGTCCGTGTTAAAACGGAGTAGCTCGAGTGACTGACTG FutX CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGCGTCTGATCAAGATGATCGGCGGC 284 CTGGGTAACCAGATGTTTATCTACGCGTTCTACCTGAAAATGAAACACCATTACCCGGAT ACGAACATCGATCTGTCTGACATGGTTCATTATAAAGTTCACAACGGTTATGAGATGAAC CGTATCTTTGACCTGAGCCAGACTGAATTTTGCATCAACCGTACCCTGAAAAAAATCCTG GAGTTCCTGTTCTTCAAAAAAATCTACGAACGTCGCCAGGACCCGTCTACTCTGTATCCA TACGAAAAACGTTATTTTTGGCCGCTGCTGTACTTTAAAGGTTTCTACCAGTCTGAACGC TTCTTCTTCGATATCAAAGACGACGTTCGTAAAGCCTTCTCTTTTAACCTGAACATCGCT AACCCGGAAAGCCTGGAACTGCTGAAACAGATCGAAGTTGACGACCAAGCTGTTTCTATC CACATCCGCCGTGGTGACTACCTGCTGCCGCGTCACTGGGCAAACACGGGTTCCGTGTGC CAGCTGCCGTATTACAAGAACGCGATCGCGGAAATGGAGAACCGTATTACTGGCCCGAGC TACTACGTGTTCTCTGATGATATCTCTTGGGTTAAAGAAAACATCCCGCTGAAGAAAGCG GTCTACGTGACGTGGAACAAGGGCGAAGACAGCTGGCAGGATATGATGCTGATGAGCCAC TGTCGTCACCACATTATCTGTAATTCTACGTTCTCCTGGTGGGGTGCTTGGCTGAACCCA CGTAAAGAGAAAATCGTCATCGCGCCGTGTCGCTGGTTCCAGCATAAAGAAACCCCGGAC ATGTACCCGAAAGAATGGATCAAAGTACCGATTAACTAGCTCGAGTGACTGACTG FutZ CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGTATTCTTGCCTGTCTGGTGGCCTG 285 GGTAACCAAATGTTTCAATACGCAGCAGCGTATATCCTGAAGCAGTATTTTCAGTCTACC ACTCTGGTCCTGGATGATAGCTATTACTATTCCCAGCCGAAACGTGATACCGTTCGTAGC CTGGAACTGAATCAGTTCAACATCTCTTATGATCGTTTTAGCTTCGCGGATGAAAAAGAG AAGATCAAACTGCTGCGCAAATTCAAACGTAACCCGTTCCCTAAACAGATTTCCGAGATC CTGTCTATTGCGCTGTTCGGCAAATACGCGCTGTCCGACCGTGCATTTTACACCTTCGAA ACTATCAAAAACATCGACAAAGCGTGCCTGTTCTCCTTTTACCAGGACGCCGATCTGCTG AATAAATATAAGCAGCTGATCCTGCCGCTGTTCGAACTGCGCGATGACCTGCTGGATATC TGCAAGAACCTGGAACTGTATTCCCTGATCCAACGCAGCAACAATACCACTGCACTGCAT ATCCGCCGTGGCGACTACGTGACCAACCAGCACGCCGCGAAATACCACGGCGTGCTGGAC ATCAGCTACTATAACCACGCAATGGAATACGTGGAACGTGAACGCGGCAAACAGAACTTC ATTATCTTCAGCGATGATGTACGTTGGGCACAGAAAGCGTTTCTGGAGAACGATAATTGC TACGTGATTAACAACTCCGACTACGATTTCTCTGCGATCGATATGTATCTGATGTCTCTG TGCAAAAACAACATCATCGCAAATTCCACCTACTCCTGGTGGGGTGCGTGGCTGAACAAA TACGAGGACAAACTGGTTATCTCTCCGAAACAATGGTTTCTGGGTAACAACGAAACCTCT CTGCGTAACGCGTCTTGGATCACCCTGTAGCTCGAGTGACTGACTG FutZA CAGTCAGTCAGAATTCAAGAAGGAGATATACATATGCGTCTGATCAAGATGACCGGTGGC 286 CTGGGTAACCAGATGTTCATCTACGCGTTTTATCTGCGTATGAAAAAACGTTATCCGAAA GTTCGTATTGATCTGTCTGATATGGTTCATTATCACGTTCACCACGGCTATGAAATGCAC CGTGTTTTCAATCTGCCGCACACCGAATTTTGCATCAACCAGCCGCTGAAAAAAGTGATC GAGTTCCTGTTTTTCAAAAAGATTTACGAACGTAAACAGGACCCTAATTCTCTGCGTGCA TTCGAGAAGAAGTATCTGTGGCCGCTGCTGTACTTCAAAGGTTTCTATCAATCTGAGCGC TTCTTTGCTGACATCAAAGACGAGGTTCGTAAAGCATTCACCTTTGACTCTTCTAAAGTG AACGCTCGCTCTGCCGAACTGCTGCGTCGCCTGGATGCCGATGCTAACGCGGTTAGCCTG CACATTCGTCGCGGTGACTATCTACAGCCGCAGCATTGGGCTACCACTGGTTCTGTCTGC CAGCTGCCGTACTACCAGAACGCGATCGCTGAAATGAACCGTCGCGTTGCTGCCCCGAGC TACTACGTTTTCAGCGATGACATCGCGTGGGTGAAGGAAAACATCCCTCTACAGAACGCA GTGTACATCGACTGGAATAAAGGCGAAGAAAGCTGGCAGGATATGATGCTGATGAGCCAC TGCCGCCACCACATTATCTGTAACAGCACCTTCTCTTGGTGGGGCGCGTGGCTGGACCCG CACGAGGACAAAATTGTAATCGTTCCGAATCGTTGGTTCCAGCATTGCGAAACTCCTAAC ATCTATCCGGCAGGCTGGGTGAAAGTTGCGATTAATTAGCTCGAGTGACTGACTG

In any of the methods described herein, the α(1,2) fucosyltransferase genes or gene products may be variants or functional fragments thereof. A variant of any of genes or gene products disclosed herein may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid or amino acid sequences described herein.

Variants as disclosed herein also include homolog, orthologs, or paralogs of the genes or gene products described herein that retain the same biological function as the genes or gene products specified herein. These variants can be used interchangeably with the genes recited in these methods. Such variants may demonstrate a percentage of homology or identity, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity conserved domains important for biological function, preferably in a functional domain, e.g. catalytic domain.

The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared, or the length of a particular fragment or functional domain thereof.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Percent identity is determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17, 3389-402). For the PSI-BLAST search, the following exemplary parameters are employed: (1) Expect threshold was 10; (2) Gap cost was Existence:11 and Extensional; (3) The Matrix employed was BLOSUM62; (4) The filter for low complexity regions was “on”.

Changes can be introduced by mutation into the nucleic acid sequence or amino acid sequence of any of the genes or gene products described herein, leading to changes in the amino acid sequence of the encoded protein or enzyme, without altering the functional ability of the protein or enzyme. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of any of sequences expressly disclosed herein. A “non-essential” amino acid residue is a residue at a position in the sequence that can be altered from the wild-type sequence of the polypeptide without altering the biological activity, whereas an “essential” amino acid residue is a residue at a position that is required for biological activity. For example, amino acid residues that are conserved among members of a family of proteins are not likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are poorly conserved among members of the protein family) may not be as essential for activity and thus are more likely to be amenable to alteration. Thus, another aspect of the invention pertains to nucleic acid molecules encoding the proteins or enzymes disclosed herein that contain changes in amino acid residues relative to the amino acid sequences disclosed herein that are not essential for activity (i.e., fucosyltransferase activity).

An isolated nucleic acid molecule encoding a protein essentially retaining the functional capability compared to any of the genes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations can be introduced into a nucleic acid sequence by standard techniques such that the encoded amino acid sequence is altered, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Certain amino acids have side chains with more than one classifiable characteristic. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a given polypeptide is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a given coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for given polypeptide biological activity to identify mutants that retain activity. Conversely, the invention also provides for variants with mutations that enhance or increase the endogenous biological activity. Following mutagenesis of the nucleic acid sequence, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. An increase, decrease, or elimination of a given biological activity of the variants disclosed herein can be readily measured by the ordinary person skilled in the art, i.e., by measuring the capability for mediating oligosaccharide modification, synthesis, or degradation (via detection of the products).

The present invention also provides for functional fragments of the genes or gene products described herein. A fragment, in the case of these sequences and all others provided herein, is defined as a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above.

For example, fragments of any of the proteins or enzymes disclosed herein or encoded by any of the genes disclosed herein can be 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, 10 to 100 amino acids, 50 to 100 amino acids, 75 to 125 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, 300 to 350 amino acids, 350 to 400 amino acids, 400 to 450 amino acids, or 450 to 500 amino acids. The fragments encompassed in the present invention comprise fragments that retain functional fragments. As such, the fragments preferably retain the catalytic domains that are required or are important for functional activity. Fragments can be determined or generated by using the sequence information herein, and the fragments can be tested for functional activity using standard methods known in the art. For example, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. The biological function of said fragment can be measured by measuring ability to synthesize or modify a substrate oligosaccharide, or conversely, to catabolize an oligosaccharide substrate.

Within the context of the invention, “functionally equivalent”, as used herein, refers to a gene or the resulting encoded protein variant or fragment thereof capable of exhibiting a substantially similar activity as the wild-type fucosyltransferase. Specifically, the fucosyltransferase activity refers to the ability to transfer a fucose sugar to an acceptor substrate via an alpha-(1,2)-linkage. As used herein, “substantially similar activity” refers to an activity level within 5%, 10%, 20%, 30%, 40%, or 50% of the wild-type fucosyltransferase.

To test for lactose-utilizing fucosylatransferase activity, the production of fucosylated oligossacharides (i.e., 2′-FL) is evaluated in a host organism that expresses the candidate enzyme (or syngene) and which contains both cytoplasmic GDP-fucose and lactose pools. The production of fucosylated oligosaccharides indicates that the candidate enzyme-encoding sequence functions as a lactose-utilizing α(1,2)fucosyltransferase.

Engineering of E. coli to Produce Human Milk Oligosaccharide 2′-FL

Described herein is a gene screening approach, which was used to validate the novel α (1,2) fucosyltransferases (α (1,2) FTs) for the synthesis of fucosyl-linked oligosaccharides in metabolically engineered E. coli. Of particular interest are α (1,2) FTs that are capable of the synthesis of the HMOS 2′-fucosyllactose (2′-FL). 2′-FL is the most abundant fucosylated oligosaccharide present in human milk, and this oligosaccharide provides protection to newborn infants against infectious diarrhea caused by bacterial pathogens such as Campylobacter jejuni (Ruiz-Palacios, G. M., et al. (2003). J Biol Chem 278, 14112-120; Morrow, A. L. et al. (2004). J Pediatr 145, 297-303; Newburg, D. S. et al. (2004). Glycobiology 14, 253-263). Other α (1,2) FTs of interest are those capable of synthesis of HMOS lactodifucotetraose (LDFT), laco-N-fucopentaose I (LNFI), or lacto-N-difucohexaose I (LDFH I).

The synthetic pathway of fucosyl oligosaccharides of human milk is illustrated in FIG. 1. Structurally, 2′-FL consists of a fucose molecule a 1,2 linked to the galactose portion of lactose (Fucα1-2Galβ1-4Glc). An α (1,2) FT from H. pylori strain 26695 termed FutC has been utilized to catalyze the synthesis of 2′-FL in metabolically engineered E. coli (Drouillard, S. et al. (2006). Angew Chem Int Ed Engl 45, 1778-780).

Candidate α(1,2) FTs (i.e., syngenes) were cloned by standard molecular biological techniques into an expression plasmid. This plasmid utilizes the strong leftwards promoter of bacteriophage λ (termed P_(L)) to direct expression of the candidate genes (Sanger, F. et al. (1982). J Mol Biol 162, 729-773). The promoter is controllable, e.g., a trp-cI construct is stably integrated the into the E. coli host's genome (at the ampC locus), and control is implemented by adding tryptophan to the growth media. Gradual induction of protein expression is accomplished using a temperature sensitive cI repressor. Another similar control strategy (temperature independent expression system) has been described (Mieschendahl et al., 1986, Bio/Technology 4:802-808). The plasmid also carries the E. coli rcsA gene to up-regulate GDP-fucose synthesis, a critical precursor for the synthesis of fucosyl-linked oligosaccharides. In addition, the plasmid carries a β-lactamase (bla) gene for maintaining the plasmid in host strains by ampicillin selection (for convenience in the laboratory) and a native thyA (thymidylate synthase) gene as an alternative means of selection in thyA⁻ hosts. Alternative selectable markers include the proBA genes to complement proline auxotrophy (Stein et al., (1984), J Bacteriol 158:2, 696-700 (1984) or purA to complement adenine auxotrophy (S. A. Wolfe, J. M. Smith, J Biol Chem 263, 19147-53 (1988)). To act as plasmid selectable markers each of these genes are first inactivated in the host cell chromosome, then wild type copies of the genes are provided on the plasmid. Alternatively a drug resistance gene may be used on the plasmid, e.g. beta-lactamase (this gene is already on the expression plasmid described above, thereby permitting selection with ampicillin). Ampicillin selection is well known in the art and described in standard manuals such as Maniatis et al., (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring, N.Y.

The nucleic acid sequence of such an expression plasmid, pEC2-(T7)FutX-rcsA-thyA (pG401) is provided below. The underlined sequence represents the FutX syngene, which can be readily replaced with any of the novel α(1,2) FTs described herein using standard recombinant DNA techniques.

(SEQ ID NO: 287) TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCG GAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCG TCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATG CGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAA TACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCTCCTCAAC CTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCT GATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCG ACGCGCAGTTTACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATT CTTTCCATCCCGATGATTGTCGCGGGTGTGATCATGATGGTCTGGGCATA TCGTCGCAGCCCACAGCAACACGTTTCCTGAGGAACCATGAAACAGTATT TAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAAAAACGACCGT ACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCT GCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCA TCATCCATGAACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTAT CTACACGAAAACAATGTCACCATCTGGGACGAATGGGCCGATGAAAACGG CGACCTCGGGCCAGTGTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAG ATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCAGCTGAAAAAC GACCCGGATTCGCGCCGCATTATTGTTICAGCGTGGAACGTAGGCGAACT GGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGG CAGACGGCAAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTC CTCGGCCTGCCGTTCAACATTGCCAGCTACGCGTTATTGGTGCATATGAT GGCGCAGCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCG ACACGCATCTGTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGC CGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATC CATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGC ATCCGGGCATTAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCA GAGCCGACGCCAGTGTGCGTCGGTTTTTTTACCCTCCGTTAAATTCTTCG AGACGCCTTCCCGAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGG GAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAG GGCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCC TGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAA ATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTG TTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAA CGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCTGA CCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAATGATAA CGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCAGAATAT CGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGAACTGATC AATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACATTGGCAAT TGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAATGATGAGCT TGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTCCTCATTAATA AACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCAAGGTCATCAAC GGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTCTACTCAACAGAT ACCCGGITAAACCTAGTCGGGTGTAACTACATAAATCCATAATAATCGTT GACATGGCATACCCTCACTCAATGCGTAACGATAATTCCCCTTACCTGAA TATTTCATCATGACTAAACGGAACAACATGGGTCACCTAATGCGCCACTC TCGCGATTTTTCAGGCGGACTTACTATCCCGTAAAGTGTTGTATAATTTG CCTGGAATTGTCTTAAAGTAAAGTAAATGTTGCGATATGTGAGTGAGCTT AAAACAAATATTTCGCTGCAGGAGTATCCTGGAAGATGTTCGTAGAAGCT TACTGCTCACAAGAAAAAAGGCACGTCATCTGACGTGCCTTTTTTATTTG TACTACCCTGTACGATTACTGCAGCTCGAGCTAGTTAATCGGTACTTTGA TCCATTCTTTCGGGTACATGTCCGGGGTTTCTTTATCCTGGAACCAGCGA CACGGCGCGATGACGATTTTCTCTTTACGTGGGTTCAGCCAAGCACCCCA CCAGGAGAACGTAGAATTACAGATAATGTGGTGACGACAGTGGCTCATCA GCATCATATCCTGCCAGCTGTCTTCGCCCTTGTTCCACGTCACGTAGACC GCTTTCTTCAGCGGGATGTTTTCTTTAACCCAAGAGATATCATCAGAGAA CACGTAGTAGCTCGGGCCAGTAATACGGTTCTCCATTTCCGCGATCGCGT TCTTGTAATACGGCAGCTGGCACACGGAACCCGTGTTTGCCCAGTGACGC GGCACCAGGTAGTCACCACGGCGGATGTGGATAGAAACAGCTTCGTCGTC AACTTCGATCTGTTTCAGCAGTTCCAGGCTTTCCGGGTTAGCGATGTTCA GGTTAAAAGAGAAGGCTTTACGAACGTCGTCTTTGATATCGAAGAAGAAG CGTTCAGACTGGTAGAAACCTTTAAAGTACAGCAGCGGCCAAAAATAACG TTTTTCGTATGGATACAGAGTAGACGCGTCCTGGCCACGTTCGTAGATTT TTTTGAAGAACAGGAACTCCAGGATTTTTTTCAGGGTACGGTTGATGCAA AATTCAGTCTGGCTCAGGTCAAAGATACGGTTCATCTCATAACCGTTGTG AACTTTATAATGAACCATGTCAGACAGATCGATGTTCGTATCCGGGTAAT GGTGTTTCATTTTCAGGTAGAACGCGTAGATAAACATCTGGTTACCCAGG CCGCCGATCATCTTGATCAGACGCATATGTATATCTCCTTCTTGAATTCT AAAAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTT TAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTT ATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAA ACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGAACTGGTTTTG CGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTC TCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCT GTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCC CCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAAT GCTTCGTTTCGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCC TTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCG TCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCG CCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGA ATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTG CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGC GGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAA TCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGT GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTC AGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTCCACGAACCCCC CGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGG ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGC TGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAA CAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGT CTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGA TTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTT TAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAAT GCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCC ATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTT ACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGG CTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGA AGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTG CCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA TTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTT GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCT CTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTC AACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC CGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG CTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTT CAGCATCTTTTACTTTCACCAGCGTTTCTGGGIGAGCAAAAACAGGAAGG CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACT CATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTC TCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTC GTC

The expression constructs were transformed into a host strain useful for the production of 2′-FL. Biosynthesis of 2′-FL requires the generation of an enhanced cellular pool of both lactose and GDP-fucose (FIG. 2). The wild-type Eschericia coli K12 prototrophic strain W3110 was selected as the parent background to test the ability of the candidates to catalyze 2′-FL production (Bachmann, B. J. (1972). Bacteriol Rev 36, 525-557). The particular W3110 derivative employed was one that previously had been modified by the introduction (at the ampC locus) of a tryptophan-inducible P_(t)r_(p)B cI+ repressor cassette, generating an E. coli strain known as GI724 (LaVallie, E. R. et al. (2000). Methods Enzymol 326, 322-340). Other features of GI724 include lacIq and lacPL8 promoter mutations. E. coli strain GI724 affords economical production of recombinant proteins from the phage λ P_(L) promoter following induction with low levels of exogenous tryptophan (LaVallie, E. R. et al. (1993). Biotechnology (N Y) 11, 187-193; Mieschendahl, et al. (1986). Bio/Technology 4, 802-08). Additional genetic alterations were made to this strain to promote the biosynthesis of 2′-FL. This was achieved in strain GI724 through several manipulations of the chromosome using λ Red recombineering (Court, D. L. et al. (2002). Annu Rev Genet 36, 361-388) and generalized P1 phage transduction.

First, the ability of the E. coli host strain to accumulate intracellular lactose was engineered by simultaneous deletion of the endogenous β-galactosidase gene (lacZ) and the lactose operon repressor gene (lad). During construction of this deletion, the lacIq promoter was placed immediately upstream of the lactose permease gene, lacY. The modified strain maintains its ability to transport lactose from the culture medium (via LacY), but is deleted for the wild-type copy of the lacZ (β-galactosidase) gene responsible for lactose catabolism. Therefore, an intracellular lactose pool is created when the modified strain is cultured in the presence of exogenous lactose. A schematic of the P_(lacIq) lacY⁺ chromosomal construct is shown in FIG. 12.

Genomic DNA sequence of the P_(lacIq) lacY⁺ chromosomal construct is set forth below (SEQ ID NO: 288):

CACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCG GAAGAGAGTCAAGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTC TAGAGAATAGGAACTTCGGAATAGGAACTTCGGAATAGGAACTAAGGAG GATATTCATATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGGTT TATTCTTTTTCTTTTACTTTTTTATCATGGGAGCCTACTTCCCGTTTTT CCCGATTTGGCTACATGACATCAACCATATCAGCAAAAGTGATACGGGT ATTATTTTTGCCGCTATTTCTCTGTTCTCGCTATTATTCCAACCGCTGT TTGGTCTGCTTTCTGACAAACTCGGGCTGCGCAAATACCTGCTGTGGAT TATTACCGGCATGTTAGTGATGTTTGCGCCGTTCTTTATTTTTATCTTC GGGCCACTGTTACAATACAACATTTTAGTAGGATCGATTGTTGGTGGTA TTTATCTAGGCTTTTGTTTTAACGCCGGTGCGCCAGCAGTAGAGGCATT TATTGAGAAAGTCAGCCGTCGCAGTAATTTCGAATTTGGTCGCGCGCGG ATGTTTGGCTGTGTTGGCTGGGCGCTGTGTGCCTCGATTGTCGGCATCA TGTTCACCATCAATAATCAGTTTGTTTTCTGGCTGGGCTCTGGCTGTGC ACTCATCCTCGCCGTTTTACTCTTTTTCGCCAAAACGGATGCGCCCTCT TCTGCCACGGTTGCCAATGCGGTAGGTGCCAACCATTCGGCATTTAGCC TTAAGCTGGCACTGGAACTGTTCAGACAGCCAAAACTGTGGTTTTTGTC ACTGTATGTTATTGGCGTTTCCTGCACCTACGATGTTTTTGACCAACAG TTTGCTAATTTCTTTACTTCGTTCTTTGCTACCGGTGAACAGGGTACGC GGGTATTTGGCTACGTAACGACAATGGGCGAATTACTTAACGCCTCGAT TATGTTCTTTGCGCCACTGATCATTAATCGCATCGGTGGGAAAAACGCC CTGCTGCTGGCTGGCACTATTATGTCTGTACGTATTATTGGCTCATCGT TCGCCACCTCAGCGCTGGAAGTGGTTATTCTGAAAACGCTGCATATGTT TGAAGTACCGTTCCTGCTGGTGGGCTGCTTTAAATATATTACCAGCCAG TTTGAAGTGCGTTTTTCAGCGACGATTTATCTGGTCTGTTTCTGCTTCT TTAAGCAACTGGCGATGATTTTTATGTCTGTACTGGCGGGCAATATGTA TGAAAGCATCGGTTTCCAGGGCGCTTATCTGGTGCTGGGTCTGGTGGCG CTGGGCTTCACCTTAATTTCCGTGTTCACGCTTAGCGGCCCCGGCCCGC TTTCCCTGCTGCGTCGTCAGGTGAATGAAGTCGCTTAAGCAATCAATGT CGGATGCGGCGCGAGCGCCTTATCCGACCAACATATCATAACGGAGTGA TCGCATTGTAAATTATAAAAATTGCCTGATACGCTGCGCTTATCAGGCC TACAAGTTCAGCGATCTACATTAGCCGCATCCGGCATGAACAAAGCGCA GGAACAAGCGTCGCA

Second, the ability of the host E. coli strain to synthesize colanic acid, an extracellular capsular polysaccharide, was eliminated by the deletion of the wcaJ gene, encoding the UDP-glucose lipid carrier transferase (Stevenson, G. et al. (1996). J Bacteriol 178, 4885-893). In a wcaJ null background GDP-fucose accumulates in the E. coli cytoplasm (Dumon, C. et al. (2001). Glycoconj J 18, 465-474). A schematic of the chromosomal deletion of wcaJ is shown in FIG. 13.

The sequence of the chromosomal region of E. coli bearing the ΔwcaJ::FRT mutation is set forth below (SEQ ID NO: 289):

GTTCGGTTATATCAATGTCAAAAACCTCACGCCGCTCAAGCTGGTGATCA ACTCCGGGAACGGCGCAGCGGGTCCGGTGGTGGACGCCATTGAAGCCCGC TTTAAAGCCCTCGGCGCGCCCGTGGAATTAATCAAAGTGCACAACACGCC GGACGGCAATTTCCCCAACGGTATTCCTAACCCACTACTGCCGGAATGCC GCGACGACACCCGCAATGCGGTCATCAAACACGGCGCGGATATGGGCATT GCTTTTGATGGCGATTTTGACCGCTGTTTCCTGTTTGACGAAAAAGGGCA GTTTATTGAGGGCTACTACATTGTCGGCCTGTTGGCAGAAGCATTCCTCG AAAAAAATCCCGGCGCGAAGATCATCCACGATCCACGTCTCTCCTGGAAC ACCGTTGATGTGGTGACTGCCGCAGGTGGCACGCCGGTAATGTCGAAAAC CGGACACGCCTTTATTAAAGAACGTATGCGCAAGGAAGACGCCATCTATG GTGGCGAAATGAGCGCCCACCATTACTTCCGTGATTTCGCTTACTGCGAC AGCGGCATGATCCCGTGGCTGCTGGTCGCCGAACTGGTGTGCCTGAAAGA TAAAACGCTGGGCGAACTGGTACGCGACCGGATGGCGGCGTTTCCGGCAA GCGGTGAGATCAACAGCAAACTGGCGCAACCCGTTGAGGCGATTAACCGC GTGGAACAGCATTTTAGCCGTGAGGCGCTGGCGGTGGATCGCACCGATGG CATCAGCATGACCTTTGCCGACTGGCGCTTTAACCTGCGCACCTCCAATA CCGAACCGGTGGTGCGCCTGAATGTGGAATCGCGCGGTGATGTGCCGCTG ATGGAAGCGCGAACGCGAACTCTGCTGACGTTGCTGAACGAGTAATGTCG GATCTTCCCTTACCCCACTGCGGGTAAGGGGCTAATAACAGGAACAACGA TGATTCCGGGGATCCGTCGACCTGCAGTTCGAAGTTCCTATTCTCTAGAA AGTATAGGAACTTCGAAGCAGCTCCAGCCTACAGTTAACAAAGCGGCATA TTGATATGAGCTTACGTGAAAAAACCATCAGCGGCGCGAAGTGGTCGGCG ATTGCCACGGTGATCATCATCGGCCTCGGGCTGGTGCAGATGACCGTGCT GGCGCGGATTATCGACAACCACCAGTTCGGCCTGCTTACCGTGTCGCTGG TGATTATCGCGCTGGCAGATACGCTTTCTGACTTCGGTATCGCTAACTCG ATTATTCAGCGAAAAGAAATCAGTCACCTTGAACTCACCACGTTGTACTG GCTGAACGTCGGGCTGGGGATCGTGGTGTGCGTGGCGGTGTTTTTGTTGA GTGATCTCATCGGCGACGTGCTGAATAACCCGGACCTGGCACCGTTGATT AAAACATTATCGCTGGCGTTTGTGGTAATCCCCCACGGGCAACAGTTCCG CGCGTTGATGCAAAAAGAGCTGGAGTTCAACAAAATCGGCATGATCGAAA CCAGCGCGGTGCTGGCGGGCTTCACTTGTACGGTGGTTAGCGCCCATTTC TGGCCGCTGGCGATGACCGCGATCCTCGGTTATCTGGTCAATAGTGCGGT GAGAACGCTGCTGTTTGGCTACTTTGGCCGCAAAATTTATCGCCCCGGTC TGCATTTCTCGCTGGCGTCGGTGGCACCGAACTTACGCTTTGGTGCCTGG CTGACGGCGGACAGCATCATCAACTATCTCAATACCAACCTTTCAACGCT CGTGCTGGCGCGTATTCTCGGCGCGGGCGTGGCAGGGGGATACAACCTGG CGTACAACGTGGCCGTTGTGCCACCGATGAAGCTGAACCCAATCATCACC CGCGTGTTGTTTCCGGCATTCGCCAAAATTCAGGACGATACCGAAAAGCT GCGTGTTAACTTCTACAAGCTGCTGTCGGTAGTGGGGATTATCAACTTTC CGGCGCTGCTCGGGCTAATGGTGGTGTCGAATAACTTTGTACCGCTGGTC TTTGGTGAGAAGTGGAACAGCATTATTCCGGTGCTGCAATTGCTGTGTGT GGTGGGTCTGCTGCGCTCCG

Third, the magnitude of the cytoplasmic GDP-fucose pool was enhanced by the introduction of a null mutation into the Ion gene. Lon is an ATP-dependant intracellular protease that is responsible for degrading RcsA, which is a positive transcriptional regulator of colanic acid biosynthesis in E. coli (Gottesman, S. & Stout, V. Mol Microbiol 5, 1599-1606 (1991)). In a Ion null background, RcsA is stabilized, RcsA levels increase, the genes responsible for GDP-fucose synthesis in E. coli are up-regulated, and intracellular GDP-fucose concentrations are enhanced. The lon gene was almost entirely deleted and replaced by an inserted functional, wild-type, but promoter-less E. coli lacZ⁺ gene (Δlon::(kan, lacZ⁺). λ Red recombineering was used to perform the construction. A schematic of the kan, lacZ⁺ insertion into the lon locus is shown in FIG. 14.

Genomic DNA sequence surrounding the lacZ+ insertion into the lon region in the E. coli strain is set forth below (SEQ ID NO: 290):

GTGGATGGAAGAGGTGGAAAAAGTGGTTATGGAGGAGTGGGTAATTGATG GTGAAAGGAAAGGGTTGGTGATTTATGGGAAGGGGGAAGGGGAAGAGGGA TGTGGTGAATAATTAAGGATTGGGATAGAATTAGTTAAGGAAAAAGGGGG GATTTTATGTGGGGTTTAATTTTTGGTGTATTGTGGGGGTTGAATGTGGG GGAAAGATGGGGATATAGTGAGGTAGATGTTAATAGATGGGGTGAAGGAG AGTGGTGTGATGTGATTAGGTGGGGGAAATTAAAGTAAGAGAGAGGTGTA TGATTGGGGGGATGGGTGGAGGTGGAGTTGGAAGTTGGTATTGTGTAGAA AGTATAGGAAGTTGAGAGGGGTTTTGAAGGTGAGGGTGGGGGAAGGAGTG AGGGGGGAAGGGGTGGTAAAGGAAGGGGAAGAGGTAGAAAGGGAGTGGGG AGAAAGGGTGGTGAGGGGGGATGAATGTGAGGTAGTGGGGTATGTGGAGA AGGGAAAAGGGAAGGGGAAAGAGAAAGGAGGTAGGTTGGAGTGGGGTTAG ATGGGGATAGGTAGAGTGGGGGGTTTTATGGAGAGGAAGGGAAGGGGAAT TGGGAGGTGGGGGGGGGTGTGGTAAGGTTGGGAAGGGGTGGAAAGTAAAG TGGATGGGTTTGTTGGGGGGAAGGATGTGATGGGGGAGGGGATGAAGATG TGATGAAGAGAGAGGATGAGGATGGTTTGGGATGATTGAAGAAGATGGAT TGGAGGGAGGTTGTGGGGGGGGTTGGGTGGAGAGGGTATTGGGGTATGAG TGGGGAGAAGAGAGAATGGGGTGGTGTGATGGGGGGGTGTTGGGGGTGTG AGGGGAGGGGGGGGGGGTTGTTTTTGTGAAGAGGGAGGTGTGGGGTGGGG TGAATGAAGTGGAGGAGGAGGGAGGGGGGGTATGGTGGGTGGGGAGGAGG GGGGTTGGTTGGGGAGGTGTGGTGGAGGTTGTGAGTGAAGGGGGAAGGGA GTGGGTGGTATTGGGGGAAGTGGGGGGGGAGGATGTGGTGTGATGTGAGG TTGGTGGTGGGGAGAAAGTATGGATGATGGGTGATGGAATGGGGGGGGTG GATAGGGTTGATGGGGGTAGGTGGGGATTGGAGGAGGAAGGGAAAGATGG GATGGAGGGAGGAGGTAGTGGGATGGAAGGGGGTGTTGTGGATGAGGATG ATGTGGAGGAAGAGGATGAGGGGGTGGGGGGAGGGGAAGTGTTGGGGAGG GTGAAGGGGGGATGGGGGAGGGGGAGGATGTGGTGGTGAGGGATGGGGAT GGGTGGTTGGGGAATATGATGGTGGAAAATGGGGGGTTTTGTGGATTGAT GGAGTGTGGGGGGGTGGGTGTGGGGGAGGGGTATGAGGAGATAGGGTTGG GTAGGGGTGATATTGGTGAAGAGGTTGGGGGGGAATGGGGTGAGGGGTTG GTGGTGGTTTAGGGTATGGGGGGTGGGGATTGGGAGGGGATGGGGTTGTA TGGGGTTGTTGAGGAGTTGTTGTAATAAGGGGATGTTGAAGTTGGTATTG GGAAGTTGGTATTGTGTAGAAAGTATAGGAAGTTGGAAGGAGGTGGAGGG TAGATAAAGGGGGGGGTTATTTTTGAGAGGAGAGGAAGTGGTAATGGTAG GGAGGGGGGGTGAGGTGGAATTGGGGGGATAGTGAGGGGGTGGAGGAGTG GTGGGGAGGAATGGGGATATGGAAAGGGTGGATATTGAGGGATGTGGGTT GTTGGGGGTGGAGGAGATGGGGATGGGTGGTTTGGATGAGTTGGTGTTGA GTGTAGGGGGTGATGTTGAAGTGGAAGTGGGGGGGGGAGTGGTGTGGGGG ATAATTGAATTGGGGGGTGGGGGAGGGGAGAGGGTTTTGGGTGGGGAAGA GGTAGGGGGTATAGATGTTGAGAATGGGAGATGGGAGGGGTGAAAAGAGG GGGGAGTAAGGGGGTGGGGATAGTTTTGTTGGGGGGGTAATGGGAGGGAG TTTAGGGGGTGTGGTAGGTGGGGGAGGTGGGAGTTGAGGGGAATGGGGGG GGGATGGGGTGTATGGGTGGGGAGTTGAAGATGAAGGGTAATGGGGATTT GAGGAGTAGGATGAATGGGGTAGGTTTTGGGGGTGATAAATAAGGTTTTG GGGTGATGGTGGGAGGGGTGAGGGGTGGTAATGAGGAGGGGATGAGGAAG TGTATGTGGGGTGGAGTGGAAGAAGGGTGGTTGGGGGTGGTAATGGGGGG GGGGGTTGGAGGGTTGGAGGGAGGGGTTAGGGTGAATGGGGGTGGGTTGA GTTAGGGGAATGTGGTTATGGAGGGGTGGAGGGGTGAAGTGATGGGGGAG GGGGGTGAGGAGTTGTTTTTTATGGGGAATGGAGATGTGTGAAAGAAAGG GTGAGTGGGGGTTAAATTGGGAAGGGTTATTAGGGAGGTGGATGGAAAAA TGGATTTGGGTGGTGGTGAGATGGGGGATGGGGTGGGAGGGGGGGGGGAG GGTGAGAGTGAGGTTTTGGGGGAGAGGGGAGTGGTGGGAGGGGGTGATGT GGGGGGGTTGTGAGGATGGGGTGGGGTTGGGTTGGAGTAGGGGTAGTGTG AGGGAGAGTTGGGGGGGGGTGTGGGGGTGGGGTAGTTGAGGGAGTTGAAT GAAGTGTTTAGGTTGTGGAGGGAGATGGAGAGGGAGTTGAGGGGTTGGGA GGGGGTTAGGATGGAGGGGGAGGATGGAGTGGAGGAGGTGGTTATGGGTA TGAGGGAAGAGGTATTGGGTGGTGAGTTGGATGGTTTGGGGGGATAAAGG GAAGTGGAAAAAGTGGTGGTGGTGTTTTGGTTGGGTGAGGGGTGGATGGG GGGTGGGGTGGGGAAAGAGGAGAGGGTTGATAGAGAAGTGGGGATGGTTG GGGGTATGGGGAAAATGAGGGGGGTAAGGGGAGGAGGGGTTGGGGTTTTG ATGATATTTAATGAGGGAGTGATGGAGGGAGTGGGAGAGGAAGGGGGGGT GTAAAGGGGGATAGTGAGGAAAGGGGTGGGAGTATTTAGGGAAAGGGGGA AGAGTGTTAGGGATGGGGTGGGGGTATTGGGAAAGGATGAGGGGGGGGGT GTGTGGAGGTAGGGAAAGGGATTTTTTGATGGAGGATTTGGGGAGAGGGG GGAAGGGGTGGTGTTGATGGAGGGGGGGGTAGATGGGGGAAATAATATGG GTGGGGGTGGTGTGGGGTGGGGGGGGTTGATAGTGGAGGGGGGGGGAAGG ATGGAGAGATTTGATGGAGGGATAGAGGGGGTGGTGATTAGGGGGGTGGG GTGATTGATTGGGGAGGGAGGAGATGATGAGAGTGGGGTGATTAGGATGG GGGTGGAGGATTGGGGTTAGGGGTTGGGTGATGGGGGGTAGGGAGGGGGG ATGATGGGTGAGAGGATTGATTGGGAGGATGGGGTGGGTTTGAATATTGG GTTGATGGAGGAGATAGAGGGGGTAGGGGTGGGAGAGGGTGTAGGAGAGG GGATGGTTGGGATAATGGGAAGAGGGGAGGGGGTTAAAGTTGTTGTGGTT GATGAGGAGGATATGGTGGAGGATGGTGTGGTGATGGATGAGGTGAGGAT GGAGAGGATGATGGTGGTGAGGGTTAAGGGGTGGAATGAGGAAGGGGTTG GGGTTGAGGAGGAGGAGAGGATTTTGAATGGGGAGGTGGGGGAAAGGGAG ATGGGAGGGTTGTGGTTGAATGAGGGTGGGGTGGGGGGTGTGGAGTTGAA GGAGGGGAGGATAGAGATTGGGGATTTGGGGGGTGGAGAGTTTGGGGTTT TGGAGGTTGAGAGGTAGTGTGAGGGGATGGGGATAAGGAGGAGGGTGATG GATAATTTGAGGGGGGAAAGGGGGGGTGGGGGTGGGGAGGTGGGTTTGAG GGTGGGATAAAGAAAGTGTTAGGGGTAGGTAGTGAGGGAAGTGGGGGGAG ATGTGAAGTTGAGGGTGGAGTAGAGGGGGGGTGAAATGATGATTAAAGGG AGTGGGAAGATGGAAATGGGTGATTTGTGTAGTGGGTTTATGGAGGAAGG AGAGGTGAGGGAAAATGGGGGTGATGGGGGAGATATGGTGATGTTGGAGA TAAGTGGGGTGAGTGGAGGGGAGGAGGATGAGGGGGAGGGGGTTTTGTGG GGGGGGTAAAAATGGGGTGAGGTGAAATTGAGAGGGGAAAGGAGTGTGGT GGGGGTAAGGGAGGGAGGGGGGGTTGGAGGAGAGATGAAAGGGGGAGTTA AGGGGATGAAAAATAATTGGGGTGTGGGGTTGGTGTAGGGAGGTTTGATG AAGATTAAATGTGAGGGAGTAAGAAGGGGTGGGATTGTGGGTGGGAAGAA AGGGGGGATTGAGGGTAATGGGATAGGTGAGGTTGGTGTAGATGGGGGGA TGGTAAGGGTGGATGTGGGAGTTTGAGGGGAGGAGGAGAGTATGGGGGTG AGGAAGATGGGAGGGAGGGAGGTTTGGGGGAGGGGTTGTGGTGGGGGAAA GGAGGGAAAGGGGGATTGGGGATTGAGGGTGGGGAAGTGTTGGGAAGGGG GATGGGTGGGGGGGTGTTGGGTATTAGGGGAGGTGGGGAAAGGGGGATGT GGTGGAAGGGGATTAAGTTGGGTAAGGGGAGGGTTTTGGGAGTGAGGAGG TTGTAAAAGGAGGGGGAGTGAATGGGTAATGATGGTGATAGTAGGTTTGG TGAGGTTGTGAGTGGAAAATAGTGAGGTGGGGGAAAATGGAGTAATAAAA AGAGGGGTGGGAGGGTAATTGGGGGTTGGGAGGGTTTTTTTGTGTGGGTA AGTTAGATGGGGGATGGGGGTTGGGGTTATTAAGGGGTGTTGTAAGGGGA TGGGTGGGGTGATATAAGTGGTGGGGGTTGGTAGGTTGAAGGATTGAAGT GGGATATAAATTATAAAGAGGAAGAGAAGAGTGAATAAATGTGAATTGAT GGAGAAGATTGGTGGAGGGGGTGATATGTGTAAAGGTGGGGGTGGGGGTG GGTTAGATGGTATTATTGGTTGGGTAAGTGAATGTGTGAAAGAAGG

Fourth, a thyA (thymidylate synthase) mutation was introduced into the strain by P1 transduction. In the absence of exogenous thymidine, thyA strains are unable to make DNA and die. The defect can be complemented in trans by supplying a wild-type thyA gene on a multicopy plasmid (Belfort, M., Maley, G. F., and Maley, F. (1983). Proc Natl Acad Sci USA 80, 1858-861). This complementation was used here as a means of plasmid maintenance.

An additional modification that is useful for increasing the cytoplasmic pool of free lactose (and hence the final yield of 2′-FL) is the incorporation of a lacA mutation. LacA is a lactose acetyltransferase that is only active when high levels of lactose accumulate in the E. coli cytoplasm. High intracellular osmolarity (e.g., caused by a high intracellular lactose pool) can inhibit bacterial growth, and E. coli has evolved a mechanism for protecting itself from high intra cellular osmlarity caused by lactose by “tagging” excess intracellular lactose with an acetyl group using LacA, and then actively expelling the acetyl-lactose from the cell (Danchin, A. Bioessays 31, 769-773 (2009)). Production of acetyl-lactose in E. coli engineered to produce 2′-FL or other human milk oligosaccharides is therefore undesirable: it reduces overall yield. Moreover, acetyl-lactose is a side product that complicates oligosaccharide purification schemes. The incorporation of a lacA mutation resolves these problems. Sub-optimal production of fucosylated oligosaccharides occurs in strains lacking either or both of the mutations in the colanic acid pathway and the Ion protease. Diversion of lactose into a side product (acetyl-lactose) occurs in strains that do not contain the lacA mutation. A schematic of the lacA deletion and corresponding genomic sequence is provided above (SEQ ID NO: 288).

The strain used to test the different α(1,2) FT candidates incorporates all the above genetic modifications and has the following genotype:

ΔampC::P_(trp) ^(B)cI, Δ(lacI-lacZ)::FRT, P_(lacIq)lacY⁺, ΔwcaJ::FRT, thyA::Tn10, Δlon:(npt3, lacZ⁺), ΔlacA

The E. coli strains harboring the different α(1,2) FT candidate expression plasmids were analyzed. Strains were grown in selective media (lacking thymidine) to early exponential phase. Lactose was then added to a final concentration of 0.5%, and tryptophan (200 μM) was added to induce expression of each candidate α(1,2) FT from the P_(L) promoter. At the end of the induction period (˜24 h) equivalent OD 600 units of each strain and the culture supernatant was harvested. Lysates were prepared and analyzed for the presence of 2′-FL by thin layer chromatography (TLC).

A map of plasmid pG217 is shown in FIG. 11, which carries the B. vulgatus FutN. The sequence of plasmid pG217 is set forth below (SEQ ID NO: 291):

TCTAGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACA CCATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTG TAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGG GCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAA AAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGAT TTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGG CGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGG TGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACA TTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGT ATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGG GCTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGC TGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCA GAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGA ATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATT CTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGG TTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAA AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG CTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT TCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTA ACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGA TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCT ACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATT AAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG TGTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGAT CTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATG ATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAA CCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTAT CCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGT AGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGG CATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAA AAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTT GGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTC TTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTAC TCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTC TTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTG AAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAAT GTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA AAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAAC CTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCG GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTC ACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGG CGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGG CATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAA TACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCTCCTC AACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTT GTTCCTGATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCC GCCAGCCCGACGCGCAGTTTACCGGTGCCTGGGTGCAGTACATCAGC ATGGGGCAAATTCTTTCCATCCCGATGATTGTCGCGGGTGTGATCAT GATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTGAG GAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAA GGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCCATTTT TGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGGTGA CAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTGG TTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGT CACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAG TGTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCAT ATTGACCAGATCACTACGGTACTGAACCAGCTGAAAAAcGACCCGGA TTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATA AAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCA GACGGCAAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTT CCTCGGCCTGCCGTTCAACATTGCCAGCTACGCGTTATTGGTGCATA TGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGACC GGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCT GCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAAC GTAAACCCGAATCCATCTTCGACTACCGTTTCGAAGACTTTGAGATT GAAGGCTACGATCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTA ATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGTCGGTTTTT TTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAGGCGCCATTC GCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCT CTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGA TTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAAC GACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGGGCATCAGGACGG TATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCCTGACTGGTGG GAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAAATACCAT TAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTGTTTA TTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAAC GGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCT GACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAAT GATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTT CAGAATATCGCCAAGGATATCGTcGAGAGATTCCGGTTTAATCGATT TAGAACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAA TGAACATTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATG TTGATTAATGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGA TGAAACAGTCCTCATTAATAAACACCACTGAAGGGCGCTGTGAATCA CAAGCTATGGCAAGGTCATCAACGGTTTCAATGTCGTTGATTTCTCT TTTTTTAACCCCTCTACTCAACAGATACCCGGTTAAACCTAGTCGGG TGTAACTACATAAATCCATAATAATCGTTGACATGGCATACCCTCAC TCAATGCGTAACGATAATTCCCCTTACCTGAATATTTCATCATGACT AAACGGAACAACATGGGTCACCTAATGCGCCACTCTCGCGATTTTTC AGGCGGACTTACTATCCCGTAAAGTGTTGTATAATTTGCCTGGAATT GTCTTAAAGTAAAGTAAATGTTGCGATATGTGAGTGAGCTTAAAACA AATATTTCGCTGCAGGAGTATCCTGGAAGATGTTCGTAGAAGCTTAC TGCTCACAAGAAAAAAGGCACGTCATCTGACGTGCCTTTTTTATTTG TACTACCCTGTACGATTACTGCAGCTCGAGTTAGGATACCGGCACTT TGATCCAACCAGTCGGGTAGATATCCGGTGCTTCGGAGTGCTGGAAC CAACGGCTCGGCACAATAACAGTCTTATCCATATTAGGGTTCAGCCA GGCACCCCACCAAGAAAACGTGCTGTTAcAAATGATGTGATGTTTGC AATGAGACATCAGCATCATATCCTGCCAGGAGTCTTCATCAGTGTTC CAGTCAATATAAACCGCATTCTGCAGTGGCAGATTTTCTTTAACCCA CGCGATATCGTCGGAGAAGATATAGTAAGATGGGCTAGCAACACGAC GGGACATTTCCGCGATAGCATTCTGGTAATACGGCAGCTGGCACACG GAACCGGTAGTAGCCCAGTGTTTCGGCTGCAGATAGTCACCACGACG AATGTGCAGGGAAACCGCGTTTTCATCTTTGTCCAGGATTTCCAGCA TGTTCAGGCTGCGGGAATTTGCTTTGTTCTTATCAAAGGTGAAGGAT TCACGCACTTCGTCTTTGATATCAGCGAAGAAACGCTCGCTCTGATA GAAACCTTTAAAGTACAGCAGCGGCCAGAAATACTTCTTCTCGAACG CACGCAGAGAGTTCGGCGCCTGCTTGCGTTCGTAGATTTTTTTAAAA AACAGGAATTCGATAACTTTTTTCAGCGGTTGGTTGATGCAGAATTC GGTGTGCGGCAGGTTGAACACGCGGTGCATTTCGTAACCGTAATGGA CTTTGTAATGCATCATGTCGCTCAGGTCGATACGGACCTTCGGGTAA TACTTTTTCATACGCAGATAGAAAGCATAGATAAACATCTGGTTGCC CAGACCGCCAGTCACTTTGATCAGACGCATTATATCTCCTTCTTG

Fucosylated oligosaccharides produced by metabolically engineered E. coli cells are purified from culture broth post-fermentation. An exemplary procedure comprises five steps. (1) Clarification: Fermentation broth is harvested and cells removed by sedimentation in a preparative centrifuge at 6000×g for 30 min. Each bioreactor run yields about 5-7 L of partially clarified supernatant. (2) Product capture on coarse carbon: A column packed with coarse carbon (Calgon 12×40 TR) of ˜1000 ml volume (dimension 5 cm diameter×60 cm length) is equilibrated with 1 column volume (CV) of water and loaded with clarified culture supernatant at a flow rate of 40 ml/min. This column has a total capacity of about 120 g of sugar. Following loading and sugar capture, the column is washed with 1.5 CV of water, then eluted with 2.5 CV of 50% ethanol or 25% isopropanol (lower concentrations of ethanol at this step (25-30%) may be sufficient for product elution.) This solvent elution step releases about 95% of the total bound sugars on the column and a small portion of the color bodies. In this first step capture of the maximal amount of sugar is the primary objective. Resolution of contaminants is not an objective. (3) Evaporation: A volume of 2.5 L of ethanol or isopropanol eluate from the capture column is rotary-evaporated at 56° C. and a sugar syrup in water is generated. Alternative methods that could be used for this step include lyophilization or spray-drying. (4) Flash chromatography on fine carbon and ion exchange media: A column (GE Healthcare HiScale50/40, 5×40 cm, max pressure 20 bar) connected to a Biotage Isolera One FLASH Chromatography System is packed with 750 ml of a Darco Activated Carbon G60 (100-mesh): Celite 535 (coarse) 1:1 mixture (both column packings were obtained from Sigma). The column is equilibrated with 5 CV of water and loaded with sugar from step 3 (10-50 g, depending on the ratio of 2′-FL to contaminating lactose), using either a celite loading cartridge or direct injection. The column is connected to an evaporative light scattering (ELSD) detector to detect peaks of eluting sugars during the chromatography. A four-step gradient of isopropanol, ethanol or methanol is run in order to separate 2′-FL from monosaccharides (if present), lactose and color bodies. Fractions corresponding to sugar peaks are collected automatically in 120-ml bottles, pooled and directed to step 5. In certain purification runs from longer-than-normal fermentations, passage of the 2′-FL-containing fraction through anion-exchange and cation exchange columns can remove excess protein/DNA/caramel body contaminants. Resins tested successfully for this purpose are Dowex 22.

The gene screening approach described herein was successfully utilized to identify new α(1,2) FTs for the efficient biosynthesis of 2′-FL in metabolically engineered E. coli host strains. The results of the screen are summarized in Table 1.

Production Host Strains

E. coli K-12 is a well-studied bacterium which has been the subject of extensive research in microbial physiology and genetics and commercially exploited for a variety of industrial uses. The natural habitat of the parent species, E. coli, is the large bowel of mammals. E. coli K-12 has a history of safe use, and its derivatives are used in a large number of industrial applications, including the production of chemicals and drugs for human administration and consumption. E. coli K-12 was originally isolated from a convalescent diphtheria patient in 1922. Because it lacks virulence characteristics, grows readily on common laboratory media, and has been used extensively for microbial physiology and genetics research, it has become the standard bacteriological strain used in microbiological research, teaching, and production of products for industry and medicine. E. coli K-12 is now considered an enfeebled organism as a result of being maintained in the laboratory environment for over 70 years. As a result, K-12 strains are unable to colonize the intestines of humans and other animals under normal conditions. Additional information on this well known strain is available at http://epa.gov/oppt/biotech/pubs/fra/fra004.htm. In addition to E. coli K12, other bacterial strains are used as production host strains, e.g., a variety of bacterial species may be used in the oligosaccharide biosynthesis methods, e.g., Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus may be modified using the methods of this invention, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also included as part of this invention are strains, modified as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).

Suitable host strains are amenable to genetic manipulation, e.g., they maintain expression constructs, accumulate precursors of the desired end product, e.g., they maintain pools of lactose and GDP-fucose, and accumulate endproduct, e.g., 2′-FL. Such strains grow well on defined minimal media that contains simple salts and generally a single carbon source. The strains engineered as described above to produce the desired fucosylated oligosaccharide(s) are grown in a minimal media. An exemplary minimal medium used in a bioreactor, minimal “FERM” medium, is detailed below.

Ferm (10 liters): Minimal medium comprising:

40 g (NH₄)₂HPO₄

100 g KH₂PO₄

10 g MgSO₄.7H₂O

40 g NaOH

1× Trace elements:

1.3 g NTA (nitrilotriacetic acid)

0.5 g FeSO₄.7H₂O

0.09 g MnCl₂.4H₂O

0.09 g ZnSO₄.7H₂O

0.01 g CoCl₂.6H₂O

0.01 g CuCl₂.2H₂O

0.02 g H₃BO₃

0.01 g Na₂MoO₄.2H₂O (pH 6.8)

Water to 10 liters

DF204 antifoam (0.1 ml/L)

150 g glycerol (initial batch growth), followed by fed batch mode with a 90% glycerol-1% MgSO₄-1× trace elements feed, at various rates for various times.

A suitable production host strain is one that is not the same bacterial strain as the source bacterial strain from which the fucosyltransferase-encoding nucleic acid sequence was identified.

Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and a fucosylated oligosaccharide is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. The fucosylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacteria are used directly in such products.

EXAMPLES Example 1: Identification of Novel α(1,2) Fucosyltransferases

To identify additional novel α(1,2)fucosyltransferases, a multiple sequence alignment query was generated using the alignment algorithm of the CLCbio Main Workbench package, version 6.9 (CLCbio, 10 Rogers Street #101, Cambridge, Mass. 02142, USA) using four previously identified lactose-utilizing α(1,2)fucosyltransferase protein sequences: H. pylori futC (SEQ ID NO: 1), H. mustelae FutL (SEQ ID NO: 2), Bacteroides vulgatus futN (SEQ ID NO: 3), and E. coli 0126 wbgL (SEQ ID NO: 4). This sequence alignment and percentages of sequence identity between the four previously identified lactose-utilizing α(1,2)fucosyltransferase protein sequences is shown in FIG. 3. An iterative PSI-BLAST was performed, using the FASTA-formatted multiple sequence alignment as the query, and the NCBI PSI-BLAST program run on a local copy of NCBI BLAST+ version 2.2.29. An initial position-specific scoring matrix file (.pssm) was generated by PSI-BLAST, which was then used to adjust the score of iterative homology search runs. The process is iterated to generate an even larger group of candidates, and the results of each run were used to further refine the matrix.

A portion of the initial position-specific scoring matrix file used is shown below:

Last position-specific scoring matrix computed A R N D C Q E G H I L K M F P S T W Y V 1 M −1 −1 −2 −3 −2 0 −2 −3 −2 1 2 −1 6 0 −3 −2 −1 −2 −1 1 2 A 2 −2 0 4 −2 −1 1 −1 −1 −2 −3 −1 −2 −3 −1 1 −1 −3 −3 −1 3 F −2 −3 −3 −4 −3 −3 −3 −3 −1 0 0 −3 0 7 −4 −3 −2 1 3 −1 4 K 0 3 0 −1 −2 1 0 −1 −1 −3 −3 3 −2 −3 −1 2 0 −3 −2 −2 5 V −1 −3 −3 −4 −1 −3 −3 −4 −3 4 2 −3 1 0 −3 −2 −1 −3 −1 3 6 V −1 −3 −3 −3 −1 −3 −3 −4 −3 4 1 −3 1 −1 −3 −2 0 −3 −1 3 7 Q −1 4 0 −1 −3 4 1 −2 0 −3 −2 3 −1 −3 −2 0 −1 −3 −2 −3 8 I −1 −3 −3 −4 −1 −2 −3 −4 −3 3 2 −3 1 0 −3 −2 −1 −3 −1 3 9 C −1 −1 0 −1 5 3 0 −2 4 −2 −2 0 −1 −2 −2 0 2 −2 −1 −1 10 G 0 −3 −1 −1 −3 −2 −2 6 −2 −4 −4 −2 −3 −3 −2 0 −2 −3 −3 −3 11 G 0 −3 −1 −1 −3 −2 −2 6 −2 −4 −4 −2 −3 −3 −2 0 −2 −3 −3 −3 12 L −2 −2 −4 −4 −1 −2 −3 −4 −3 2 4 −3 2 0 −3 −3 −1 −2 −1 1 13 G 0 −3 −1 −1 −3 −2 −2 6 −2 −4 −4 −2 −3 −3 −2 0 −2 −3 −3 −3 14 N −2 −1 6 1 −3 0 0 −1 1 −4 −4 0 −2 −3 −2 1 0 −4 −2 −3 15 Q −1 1 0 0 −3 6 2 −2 0 −3 −2 1 −1 −3 −1 0 −1 −2 −2 −2 16 M −1 −2 −3 −4 −2 −1 −2 −3 −2 1 3 −2 5 0 −3 −2 −1 −2 −1 1 17 F −2 −3 −3 −4 −3 −3 −4 −3 −1 0 0 −3 0 7 −4 −3 −2 1 3 −1 18 Q −1 0 −1 −1 −3 5 1 −2 0 1 −1 1 0 −2 −2 −1 −1 −2 −2 0 19 Y −2 −2 −3 −3 −3 −2 −3 −3 1 −1 −1 −2 −1 5 −3 −2 −2 2 6 −1 20 A 4 −1 −1 −1 −1 −1 −1 0 −2 −2 −2 −1 −1 −2 −1 2 0 −3 −2 −1 21 F −2 −3 −3 −4 −3 −3 −4 −3 −1 0 0 −3 0 7 −4 −3 −2 1 3 −1 22 A 3 −2 −1 −2 −1 −1 −1 4 −2 −2 −2 −1 −2 −3 −1 1 −1 −3 −2 −1 23 K −1 0 −1 −2 −3 0 −1 −3 1 −2 −2 3 −1 2 −2 −1 −1 1 6 −2 24 S 2 −1 −1 −2 −1 −1 −1 −1 −2 −1 1 −1 0 −1 −1 3 0 −3 −2 0 25 L −2 3 −2 −3 −2 −1 −2 −3 −2 1 3 0 1 0 −3 −2 −1 −2 −1 0 26 Q 0 0 0 −1 −2 4 1 −2 −1 −1 0 0 3 −2 −2 2 0 −2 −2 −1 27 K −1 2 0 −1 −3 1 0 −2 −1 −2 −2 4 −1 −3 −1 0 2 −3 −2 −2 28 H −1 0 0 −2 −3 0 0 −2 6 1 −1 2 −1 −1 −2 −1 −1 −3 0 0 29 S −1 −1 3 −1 −2 −1 −1 −2 0 −1 1 −1 0 1 −2 1 0 0 4 −1 30 H −1 −1 4 0 −3 −1 −1 3 0 −3 −3 −1 −2 0 −3 0 −1 −1 4 −3 31 T −1 −2 −1 −2 −2 −1 −2 −2 −2 1 −1 −1 −1 −2 5 0 3 −3 −2 0 32 P −1 0 −2 −1 −3 0 −1 −2 −2 −3 −3 2 −2 −4 7 −1 −1 −4 −3 −3 33 V −1 −3 −3 −4 −1 −2 −3 −4 −3 2 2 −3 1 −1 −3 −2 0 −3 −1 4 34 L −2 3 −2 −3 −2 −1 −2 −3 0 0 2 0 1 1 −3 −2 −1 0 4 −1 35 L −2 −3 −4 −4 −2 −3 −3 −4 −3 3 3 −3 1 3 −3 −3 −1 −1 1 1 36 D −2 −2 1 6 −4 0 1 −2 −1 −4 −4 −1 −3 −4 −2 0 −1 −5 −3 −4

The command line of PSI-BLAST that was used is as follows: psiblast-db<LOCAL NR database name>-max_target_seqs 2500-in_msa<MSA file in FAST format>-out<results output file>-outfmt “7sskingdoms sscinames scomnames sseqid stitle evalue length pident”-out_pssm<PSSM file output>-out_ascii_pssm<PSSM (ascii) output>-num_iterations 6-num_threads 8

This PSI-BLAST search resulted in an initial 2515 hits. There were 787 hits with greater than 22% sequence identity to FutC. 396 hits were of greater than 275 amino acids in length. Additional analysis of the hits was performed, including sorting by percentage identity to FutC, comparing the sequences by BLAST to an existing α(1,2) fucosyltransferase inventory (of known α(1,2) fucosyltransferases, to eliminate known lactose-utilizing enzymes and duplicate hits), and manual annotation of hits to identify those originating from bacteria that naturally exist in the gastrointestinal tract. An annotated list of the novel α(1,2) fucosyltransferases identified by this screen are listed in Table 1. Table 1 provides the bacterial species from which the enzyme is found, the GenBank Accession Number, GI Identification Number, amino acid sequence, and % sequence identity to FutC.

Multiple sequence alignment of the 4 known α(1,2) FTs used for the PSI-BLAST query and 12 newly identified α(1,2) FTs is shown in FIG. 4.

Example 2: Validation of Novel α(1,2) FTs

To test for lactose-utilizing fucosylatransferase activity, the production of fucosylated oligossacharides (i.e., 2′-FL) is evaluated in a host organism that expresses the candidate enzyme (i.e., syngene) and which contains both cytoplasmic GDP-fucose and lactose pools. The production of fucosylated oligosaccharides indicates that the candidate enzyme-encoding sequence functions as a lactose-utilizing α(1,2)fucosyltransferase. Of the identified hits, 12 novel α(1,2) fucosyltransferases were further analyzed for their functional capacity to produce 2′-fucosyllactose: Prevotella melaninogenica FutO, Clostridium bolteae FutP, Clostridium bolteae+13 FutP, Lachnospiraceae sp. FutQ, Methanosphaerula palustries FutR, Tannerella sp. FutS, Bacteroides caccae FutU, Butyrivibrio FutV, Prevotellaa sp. FutW, Parabacteroides johnsonii FutX, Akkermansia muciniphilia FutY, Salmonella enterica FutZ, and Bacteroides sp. FutZA.

Syngenes were constructed comprising the 12 novel α(1,2) FTs in the configuration as follows: EcoRI-T7g10 RBS-syngene-XhoI. FIG. 5A and FIG. 5B show the syngene fragments after PCR assembly and gel-purification.

The candidate α(1,2) FTs (i.e., syngenes) were cloned by standard molecular biological techniques into an exemplary expression plasmid pEC2-(T7)-Fut syngene-rcsA-thyA. This plasmid utilizes the strong leftwards promoter of bacteriophage X (termed P_(L)) to direct expression of the candidate genes (Sanger, F. et al. (1982). J Mol Biol 162, 729-773). The promoter is controllable, e.g., a trp-cI construct is stably integrated the into the E. coli host's genome (at the ampC locus), and control is implemented by adding tryptophan to the growth media. Gradual induction of protein expression is accomplished using a temperature sensitive cI repressor. Another similar control strategy (temperature independent expression system) has been described (Mieschendahl et al., 1986, Bio/Technology 4:802-808). The plasmid also carries the E. coli rcsA gene to up-regulate GDP-fucose synthesis, a critical precursor for the synthesis of fucosyl-linked oligosaccharides. In addition, the plasmid carries a β-lactamase (bla) gene for maintaining the plasmid in host strains by ampicillin selection (for convenience in the laboratory) and a native thyA (thymidylate synthase) gene as an alternative means of selection in thyA⁻ hosts.

The expression constructs were transformed into a host strain useful for the production of 2′-FL. The host strain used to test the different α(1,2) FT candidates incorporates all the above genetic modifications described above and has the following genotype:

ΔampC::P_(trp) ^(B)cI, Δ(lacI-lacZ)::FRT, P_(lacIq)lacY⁺, ΔwcaJ::FRT, thyA::Tn10, Δlon:(npt3, lacZ⁺), ΔlacA

The E. coli strains harboring the different α(1,2) FT candidate expression plasmids were analyzed. Strains were grown in selective media (lacking thymidine) to early exponential phase. Lactose was then added to a final concentration of 0.5%, and tryptophan (200 μM) was added to induce expression of each candidate α(1,2) FT from the P_(L) promoter. At the end of the induction period (˜24 h) the culture supernatants and cells were harvested. Heat extracts were prepared from whole cells and the equivalent of 0.2OD₆₀₀ units of each strain analyzed for the presence of 2′-FL by thin layer chromatography (TLC), along with 2 μl of the corresponding clarified culture supernatant for each strain.

FIG. 6 shows the oligosaccharides produced by the α(1,2) FT-expressing bacteria, as determined by TLC analysis of the culture supernatant and extracts from the bacterial cells. 2′FL was produced by exogenous expression of WbgL (used as control), FutO, FutP, FutQ, FutR, FutS, FutU, FutW, FutX, FutZ, and FutZA.

Table 4 summarizes the fucosyltransferase activity for each candidate syngene as determined by the 2′FL synthesis screen described above. 11 of the 12 candidate α(1,2) FTs were found to have lactose-utilizing fucosyltransferase activity.

TABLE 4 2′FL synthesis screen results 24 h OD 2′-FL culture 2′-FL cell syngene (induced) medium extract Escherichia coli WbgL 9.58 5 5 pG204 pEC2-WbgL-rcsA-thyA E640 Prevotella melaninogenica FutO 12.2 3 2 pG393 pEC2-(T7)FutO-rcsA-thyA E985 Clostridium bolteae FutP 10.4 1 2 pG394 pEC2-(T7)FutP-rcsA-thyA E986 Lachnospiraceae sp. FutQ 10.6 3 4 pG395 pEC2-(T7)FutQ-rcsA-thyA E987 Methanosphaerula palustris FutR 11.9 0 1 pG396 pEC2-(T7)FutR-rcsA-thyA E988 Tannerella sp. FutS 11.3 2 3 pG397 pEC2-(T7)FutS-rcsA-thyA E989 Bacteroides caccae FutU 12.1 0 2 pG398 pEC2-(T7)FutU-rcsA-thyA E990 Butyrlvibrio FutV 11.3 0 1 pG399 pEC2-(T7)FutV-rcsA-thyA E991 Prevotella sp. FutW 10.5 3 3 pG400 pEC2-(T7)FutW-rcsA-thyA E992 Parabacteroides johnsonii FutX 10.7 3 5 pG401 pEC2-(T7)FutX-rcsA-thyA E993 Akkermansia muciniphilia FutY 9.1 0 0 pG402 pEC2-(T7)FutY-rcsA-thyA E994 Salmonella enterica FutZ 11.0 0 3 pG403 pEC2-(T7)FutZ-rcsA-thyA E995 Bacteroides sp. FutZA 9.9 3 3 pG404 pEC2-(T7)FutZA-rcsA-thyA E996

Example 3: Characterization of Cultures Expressing Novel α(1,2) FTs

Further characterization of the bacterium expressing novel α(1,2) FTs FutO, FutQ, and FutX was performed. Specifically, proliferation rate and exogenous α(1,2) FT expression was examined.

Expression plasmids containing fucosyltransferases WbgL (plasmid pG204), FutN (plasmid pG217), and novel α(1,2) FTs FutO (plasmid pG393), FutQ (plasmid pG395), and FutX (pG401) were introduced into host bacterial strains. For example, the host strains utilized has the following genotype: ΔampC::P_(trp) ^(B)cI, Δ(lacI-lacZ)::FRT, P_(lacIq)lacY⁺, ΔwcaJ::FRT, thyA::Tn10, Δlon:(npt3, lacZ⁺), ΔlacA

Bacterial cultures expressing each exogenous fucosyltransferase were induced by addition of tryptophan (to induce expression of the exogenous fucosyltransferases) in the presence of lactose. Growth of the cultures was monitored by spectrophotometric readings at A600 at the following timepoints: 4 hours and 1 hour before induction, at the time of induction (time 0), and 3 hours, 7 hours, and 24 hours after induction. The results are shown in FIG. 7, and indicate that expression of the exogenous fucosyltransferase did not prevent cell proliferation. Furthermore, the growth curve for the bacterial cultures expressing the novel α(1,2) fucosyltransferases FutO, FutQ, and FutX is similar to those expressing the known α(1,2)FT enzymes WbgL and FutN.

Protein expression was also assessed for the bacterial cultures expressing each fucosyltransferase after induction. Cultures were induced as described previously, and protein lysates were prepared from the bacterial cultures at the time of induction (0 hours), 3 hours, 7 hours, and 24 hours after induction. The protein lysates were run on an SDS-PAGE gel and stained to examine the distribution of proteins at each time point. As shown in FIG. 8, induction at 7 hours and 24 hours showed increases in a protein band at around 20-28 kDa for bacterial cultures expressing exogenous FutN, FutO, and FutX. These results indicate that induction results in significant expression of the exogenous fucosyltransferases.

Finally, additional TLC analysis to assess the efficiency and yield of 2′FL production in bacterial cultures expressing novel α(1,2) FTs FutO, FutQ, and FutX compared to known fucosyltransferases WbgL and FutN. Cultures were induced at 7 hours and 24 hours, and run out on TLC. FIG. 9A shows the level of 2′FL in the cell supernatant. The level of 2′FL found in the bacterial cells were also examined. As shown in FIG. 9B, 2′FL was produced in cell lysates from bacteria expressing the novel α(1,2) FTs FutO, FutQ, and FutX at 7 hours and 24 hours after induction.

Example 4: FutN Exhibits Increased Efficiency for Production of 2′FL

Fucosylated oligosaccharides produced by metabolically engineered E. coli cells to express B. vulgatus FutN was purified from culture broth post-fermentation.

Fermentation broth was harvested and cells were removed by sedimentation in a preparative centrifuge at 6000×g for 30 min. Each bioreactor run yields about 5-7 L of partially clarified supernatant. A column packed with coarse carbon (Calgon 12×40 TR) of ˜1000 ml volume (dimension 5 cm diameter×60 cm length) was equilibrated with 1 column volume (CV) of water and loaded with clarified culture supernatant at a flow rate of 40 ml/min. This column had a total capacity of about 120 g of sugar. Following loading and sugar capture, the column is washed with 1.5 CV of water, then was eluted with 2.5 CV of 50% ethanol or 25% isopropanol (lower concentrations of ethanol at this step (25-30%) may be sufficient for product elution.) This solvent elution step released about 95% of the total bound sugars on the column and a small portion of color bodies (caramelized sugars). A volume of 2.5 L of ethanol or isopropanol eluate from the capture column was rotary-evaporated at 56° C. and a sugar syrup in water was generated. A column (GE Healthcare HiScale50/40, 5×40 cm, max pressure 20 bar) connected to a Biotage Isolera One FLASH Chromatography System was packed with 750 ml of a Darco Activated Carbon G60 (100-mesh): Celite 535 (coarse) 1:1 mixture (both column packings were obtained from Sigma). The column was equilibrated with 5 CV of water and loaded with sugar from step 3 (10-50 g, depending on the ratio of 2′-FL to contaminating lactose), using either a celite loading cartridge or direct injection. The column was connected to an evaporative light scattering (ELSD) detector to detect peaks of eluting sugars during the chromatography. A four-step gradient of isopropanol, ethanol or methanol was run in order to separate 2′-FL from monosaccharides (if present), lactose and color bodies. Fractions corresponding to sugar peaks were collected automatically in 120-ml bottles, pooled.

The results from two fermentation runs are shown in FIG. 10A and FIG. 10B. The cultures were grown for 136 (run 36B) or 112 hours (run 37A), and the levels of 2′-FL produced was analyzed by TLC analysis. As shown in both FIG. 10A and FIG. 10B, the 2′-fucosyllactose was produced at 40 hours of culture, and production continued to increase until the end point of the fermentation process. The yield of 2′-FL produced from run 36B was 33 grams per liter. The yield of 2′-FL produced from run 37A was 36.3 grams per liter. These results indicate that expression of exogenous FutN is suitable for high yield of 2′-fucosyllactose product.

TABLE 1 % i- SEQ Bacterium Accession Gene name dentity ID names No. GI No. [bacterium] FutC Alias SEQUENCE NO Helicobacter  AAD29869.1 4808599 alpha-1,2- 98 FutC MAFKVVQICGGLGNQMFQYAFAKSLQKHSNTPVLLDITSEDWSDRKMQLELFPINLPYASAKEIAIAKMQH 1 pylori fucosyl- LPKLVRDALKCMGFDRVSQEIVFEYEPELLKPSRLTYFYGYFQDPRYFDAISPLIKQTFTLPPPPENNKNNNKKE transferase  EEYHRKLSLILAAKNSVFVHIRRGDYVGIGCQLGIDYQKKALEYMAKRVPNMELFVFCEDLEFTQNLDLGYPF [Helicobacter MDMITRNKEEEAYWDMLLMQSCQHGBANSTYSWWAAYLIENPEKIIIGPKHWLFGHENILCKEWVKIESH pylori] FEVKSQKYNA Helicobacter YP_ 291277413 alpha-1,2- 70.85 FutL MDFKIVQVHGGLGNQMFQYAFAKSLQTHLNIPVLLDTTWFDYGNRELGLHLFPIDLCICASAQQIAAAHMQ 2 mustelae;  003517185.1 fucosyl- NLPRLVRGALRRMGLGRVSKEIVFEYMPELFEPSRIAYFHGYEQDPRYFEDISPLIKQTFTLPHPTEHAEQYSR Helicobacter transferase  KLSQILAAKNSVFVHIRRGDYMRLGWQLDISYQLRAIAYMAKRVQNLELFLECEDLEFVQNLDLGYPFVDMT mustelae 12198 [Helicobacter TRDGAAHWDMMLMQSCKHGIITNSTYSWWAAYLIKNPEKIIIGPSHWIYGNENILCKDWVKIESQFETKS mustelae  12198] Bacteroides;  YP_ 150005717 glycosyl 24.83 FutN MRLIKVTGGLGNQMFIYAFYLRMKKYYPKVRIDLSDMMHYKVHYGYEMHRVFNLPHTEFCINQPLKKVIEFL 3 Bacteroides  001300461.1 transferase FFKKIYERKQAPNSLRAFEKKYFWPLLYEKGFYQSERFFADIKDEVRESFTFDKNKANSRSLNMLEILDKDENA vulgatus ATCC  family protein VSLHIRRGDYLQPKHWATTGSVCQLPYYCINAIAEMSRRVASPSYYIFSDDIAWVKENLPLQNAVYIDWNTD 8482;  [Bacteroides EDSWQDMMLMSHCKHHIICNSTFSWWGAWLNPNIVIDKTVIVPSRWFIDHSEAPDIYPTGWIKVPVS Bacteroides  vulgatus ATCC sp. 4_3_47FAA;  8482] Bacteroides  sp. 3_1_40A;  Bacteroides vulgatus PC510;  Bacteroides vulgatus CL09T03C04;  Bacteroides vulgatus dnLKV7;  Bacteroides vulgatus CAG:6 Escherichia WP_ 545259828 protein 23.13 WbgL MSIIRLQGGLGNQLFQFSFGYALSKINGTPLYFDISHYAENDDHGGYRLNNLQIPEEYLQYYTPKINNIYKLLVR 4 coli;  021554465.1 [Escherichia  GSRLYPDIFLELGFCNEFHAYGYDFEYIAQKWKSKKYIGYVVQSEHFFHKHILDLKEFFIPKNVSEQANLLAAKIL Escherichia  coli] ESQSSLSIHIRRGDYIKNKTATLTHGVCSLEYYKKALNKIRDLAMIRDVFIFSDDIFWCKENIETLLSKKYNIYYSE coli DLSQEEDLWLMSLANHHIIANSSFSWWGAYLGSSASQIVIYPTPWYDITPKNTYIPIVNHWINVDKHSSC UMEA 3065-1 Helicobacter  WP_ 491361813 predicted 36.79 FutD MGDYKIVELTCGLGNQMFQYAFAKALQKHLQVPVLLDKIWYDTQDNSTQFSLDIENVDLEYATNTQIEKAK 5  bilis; 005219731.1 protein ARVSKLPGLLIIKMFGLKKHNIAYSQSFDFHDEYLLPNDFTYFSGFFQNAKYLKGLEQELKSIFYYDSNNESNEG Helicobacter  [Helicobacter KQRLELILQAKNSIFINIRRGDYCKIGWELGMDYYKRAIQVIMDRVEEPKFFIFGATDMSFTEQFQKNLGLNE bills bills] NNSANLSEKTITQDNQHEDMFLIVICYCKHAILANSSYSEWSAYLNNDANNIVIAPTPWLLDNDNIICDDWIKI ATCC 43879 SSK Escherichia  AAO37698.1 37788088 fucosyl- 25.94 Wbs.1 MEVKIIGGLGNQMFQYATAFAIAKRTHQNLTVDISDAVKYKTHPLRLVELSCSSEEVKKAWPFEKYLFSEKIPH 6 coli transferase  FMKKGMERKHYVEKSLEYDPDIDTKSINKKIVGYFQTEKYFKEFRHELIKEFQPKTKFNSYQNELLNLIKENDTC [Escherichia SLHIRRGDYVSSKIANETHGTCSEKYFERAIDYLMNKGVINKKTLLFIFSDDIKWCRENIFFNNQICFVQGDAY coil] HVELDMLITVISKCKNNIISNSSFSWWAAWLNENKNKTVIAPSKWEKKDIKHDIIPESWVKL Vibrio  BAA33632.1 3721682 probable beta- 25.94 WbIA MIVMKISGGLGNQLFQYAVGRAIAIQYGVPLKLDVSAYKNYKLHNGYRLDQFNINADIANEDEIFHLKGSSN 7 cholerae D-galactoside  RLSRILRRLGWLKKNTYYAEKQRTIYDVSVEMQAPRYLDGYWQNEQYFSQIRAVLLQELWPNQPLSINAQA 2-alpha-L- HQIKIQQTHAVSIHVRRGDYLNHPEIGVLDIDYYKRAVDYIKEKIEATVEFVF5NDVAWCKDNENFIDSPVFIE fucosyl- DTQTEIDDLMLMCQCQHNIVANSSFSWWAAWLNSNVDKIVIAPKTWMAENPKGYKWVPDSWREI transferase [Vibrio cholerae] Bacteroides YP_099118.1 53713126 alpha-1,2- 24.58 Bft2 MIVSSLRGGLGNQMFIYAMVKAMALRNNVPFAFNETTDEANDEVYKRKLLLSYFALDLPENKKLTFDFSYGN 8 fragilis;  fucosyl- YYRRLSRNLGCHILHPSYRYICEERPPHEESRLISSKITNAFLEGYWQSEKYFLDYKQEIKEDPVIQKKLEYTSYLE Bacteroides transferase  LEEIKLLDKNAIMIGVRRYQESDVAPGGVLEDDYYKCAMDIMASKVTSPVFFCFSQDLEWVEKHLAGKYPVR fragilis NCTC [Bacteroides LISKKEDDSGTIDDMELMMHERNYIISNSSFYWWGAWLSKYDDKLVIAPGNFINKDSVPESWFKLNVR 9343;  fragilis  Bacteroides  YCH46] fragilis  YCH46;  Bacteroides fragilis HMW  615 Escherichia WP_ 486356116 protein 24.25 WbgN MSIVVARLAGGLGNQMFQYAKGYAESVERNSYLKLDLRGYKNYTLHGGFRLDKLNIDNTFVMSKKEMCIFP 9 coli;  001592236.1 [Escherichia NFIVRAINKFPKLSLCSKRFESEQYSKKINGSMKGSVEFIGEWQNERYFLEHKEKLREIFTPININLDAKELSDVI Escherichia  coli] RCTNSVSVHIRRGDYVSNVEALKIHGLCTERYYIDSIRYLKERFNNLVFFVESDDIEWCKKYKNEIESRSDDVKFI coli KTE84 EGNTQEVDMWLMSNAKYHIIANSSFSWWGAWLKNYDLGITIAPTPWEEREELNSFDPCPEKWVRIEK Prevotella YP_ 302346214 glycosyl- 31.1 FutO MKIVKILGGLGNQMFQYALYLSLQESFPKERVALDLSSFHGYHLHNGFELENIFSVTAQKASAADIMRIAYYY 10 malenino- 003814512.1 transferase  PNYLLWRIGKRFLPRRKGMCLESSSLREDESVLRQEGNRYFDGYVVQDERYFAAYREKVLKAFTEPAFKRAEN genica;  family 11 LSLLEKLDENSIALHVRRGDYVGNNLYQGICDLDYYRTAIEKMCAHVTPSLFCIFSNDITWCQQHLQPYLKAP Prevotella [Prevotella VVYVTWNTGVESYRDMQLMSCCAHNIIANSSFSWWGAWLNQNREKVVIAPKKWLNMEECHFTLPASWI melaninogenica  melaninogenica KI ATCC 25845 ATCC 25845] Clostridium WP_ 488634090 protein 29.86 FutP MFQYALYKAFEQKHIDVYADLAWYKNKSVKFELYNFGIKINVASEKDINRLSDCQADEVSRIRRKIEGKKKSEV 11 bolteae;  002570768.1 [Clostridium SEKNDSCYENDILRMDNVYLSGYWQTEKYFSNTREKLLEDYSFALVNSQVSEWEDSIRNKNSVSIHIRRGDYL CloStridium  bolteae] QGELYGGICTSLYYAEAIEYIKNARVPNAKFFVFSDDVEWVKQQEDFKGFVIVDRNEYSSALSDMYLMSLCKH bolteae 90A9;  NIIANSSFSWWAAWLNRNEEKIVIAPRRWLNGKCTPDIWCKKWIRI Clostridium bolteae 90133;  Clostridium bolteae 90B8 Lachno- WP_ 496545268 protein 29.25 FutQ MVIVQLSGGLGNQMFEYALYLSLKAKGKEVKIDDVTCYEGPGTRPRQLDVEGITYDRASREELTEMTDASM 12 spiraceae 009251343.1 [Lachnos- DALSRVRRKLTGRRTKAYRERDINFDPLVMEKDPALLEGCFQSDKYFRDCEGRVREAYRERGIESGAFPLPED bacterium piraceae YLRLEKQIEDCQSVSVHIRRGDYLDESHGGLYTGICTEAYYKEAFARMERLVPGARFFLFSNDPEVVTREHFES 3_1_57FAA_CT1 bacterium KNCVLVEGSTEDTGYMDLYLMSRCRHNIIANSSFSWWGAWLNENPEKKVIAPAKWLNGRECRDIYTERMI 3_1_ RL 57 FAA_CT1] Methano- YP_ 219852781 glycosyl 28.52 FutR MIIVRLKGGLGNQLSQYALGRKIAHLHNTELKLDTTWFTTISSDTPRTYRLNNYNIIGTIASAKEIGLIERGRAQ 13 sphaerula transferase GRGYLLSKISDLLTPMYRRTYVRERMHTFDKAILTVPDNVYLDGYWQTEKYFKDIEEILRREVTLKDEPDSINLE palustris;  002467213.1 family protein MAERIQACHSVSLHVRRGDYVSNPTTQQFHGCCSIDYYNRAISLIEEKVDDPSFFIFSDDLPWAKENLDIPGE Methano- [Methano- KTFVAHNGPEKEYCDLWLMSLCQHHIIANSSFSWWGAWLGQDAEKMVIAPRRWALSESFDTSDIIPDSWI sphaerula  sphaerula TI palustris  palustris  E1-9c E1-9c] Tannerella   WP_ 547187521 glycosyl 28.38 FutS MVRIVEIIGGLGNQMFWQYAFSLYLKNKSHIWDRLYVDIEAMKTYDRHGLELEKVFNLSLCPISNRLHRNLQK 14 sp. CAG:118 021929367.1 transferase RSFAKHFVKSLYEHSECEFDEPVYRGLRPYRYYRGYWQNEGYFVDIEMIREAFQFNVNILSKKTKAIASKMR family 11 RELSVSIHVRRGDYENLPEAKAMHGGICSLDYYHGKAIDFIRQRLDNNICFYLFSDDINWVEENLQLENRCIID [Tannerella  WNQGESDSWQDMYLMSCCRHHIIANSSFSWWAAWLNPNKWFNHTDAVGIVPGSWIKIPVF sp. CAG:118] Bacteroides WP_ 491925845 protein 28.09 FutU MKIVKILGGLGNQMFQYALFLSLKERFPHEQVMIDTSCFRNYPLHNGFEVDRIFAQKAPVASWRNILKVAYP 15 caccae  005675707.1 [Bacteroides YPNYRFWKIGKYILPKRKTMCVERKNFSFDAAVLTRKGDCYYDGYWQHEEYFCDMKETIWEARSFPEPVDG Bacteroides  caccae] RNKEIGALLQASDSASLHVRRGDYVNHPLFRGICDLDYYKRAIHYMEERVNPQLYCFSNDMAWCESHLRA caccae ATCC  LLPGKEVVYVDWNKGAESYVDMRLMSLCRHNIIANSSFSWWGAWLNRNPQKVVVAPERWMNSPIEDPV 43185 SDKWIKL Butyrivibrio  WP_ 551028636 protein 27.8 FutV VIIQLKGGLGNQMFQYALKSLKKRGKEVKIDDKTGFVNDKLRIPVLSRWGVEYDRATDEEIINLTDSKMDL 16 sp. AE2015 022772718.1 {Butyrivibrio FSRIRRKLTGRKTFRIDEESGKFNPELEKENAYLVGYWQCDKYFDDKDVVREIREAFEKKPQELMTDASSWS sp. AE2015] TLQQIECCESVSLHVRRTDYVDEEHIHIHNICTEDYYKNAIDRVKQYPSAVFFIFTDDKEWCRDHFKGPNFIV VELEEGDGTDIAEMTLMSRCKHHIIANSSFSWWAAWLNDSPEKIVIAPQKWINNRDMDDIYTERMTKIAL Prevotella  WP_ 548264264 un- 27.4 FutW MRLVKMIGGLGNQMFIYAFYLQMRKRFSNVRIDLDMMHYNVHYGYELHKVFGLPRTEFCMNQPLKKVL 17 sp. CAG:891 022481266.1 characterized EFLFFRTIVERKQHGRMEPYTCQVYWPLVYFKGFYQSERYFSEVKDEVRECFTFNPALANRSSQQMMEQIQ protein NDPQAVSIHIRRGDYLNPKHYDTIGCICQLPYYKHAVSEIKKYVSNPHFYVFSEDLDWVKANLPLENAQYIDW [Prevotella  NKGADSWQDMMLMSCCKHHIICNSTFSWWAAWLNPSVEKTVIMPEQWTSRQDSVDFVASCGRWVRV sp. CAG: 891] KTE Para- WP_ 495431188 glycosol 26.69 FutX MRLIKMIGGLGNQMFIYAFYLKMKHHYPDTNIDLSMNVHYKVHNGYEMNRIFDLSQTEFCINRTLKKILEFL 18 bacteroides 008155883.1 transferase FFKKIYERRQDPSTLYPYEKRYFWPLLYFKGFYQSERFFFDIKDDVRKAFSFNLNIANPESELLKQIEVDDQAV johnsonii;  [Para- SIHIRRGDYLLPRHWANTGSVCQLPYYKNAIAEMENRITGPSYYVFSDDISWVKENIPLKKAVYVTWNKGED Para- bacteroides SWQDMMLMSHCRHHIICNSTFSWWGAWLNPREKIVIAPCRWFQHKEPPDMYPKEWIKVPIN bacteroides  johnsonii] johnsonii CL02T1C29 Akkemansia YP_ 187735443 glycosyl 25.67 FutY MRLFGGLGNQLFQYAFLFALSRQGGKARLETSSYEHDDKRVCELHHFRVSLPIEGGPPPWAFRKSRIPACLRS 19 muciniphila; 001877555.1 transferase LFAAPKYPHFREEKRHGFDPGLAAPPRRHTYFKGYFQTEQYFLHCREQLCREFRLKTPLTPENARILEDIRSCCS Akkermansia family protein ISLHIRRTDYLSNPYLSPPPLEYYLRSMAEMEGRLAAGAPQESLRYFIFSDDIEWARQNLRPALPHVHVDIND muciniphila [Akkermansia GGTGYFDLELMRNCRHHIIANSTFSWWAAWLNEHAEKIVIAPRIWFNREEGDRYHTDDALIPGSWLRI ATCC BAA-835 muciniphila ATCC BAA-835] Salmonella WP_ 555221695 fucosyl- 25.99 FutZ MYSCLSGGLGNQMFQYAAAYILKQYFQSTTLVLDDSYYYSQPKRDTVRSLELNQFNISYDRFSFADEKEKIKLL 20 enterica; 023214330.1 transferase RKFKRNPFPKQISEILSIALFGKYALSDRAFYTFETIKNIDKACLFSFYQDADLLNKYKQLILPLFELRDDLLDICKN Salmonella [Salmonella LELYSLIQRSNNTTALHIRRGDYVTNQHAAKYHGVLDISYYNHAMEYVERERFKQNFIIFSDDVRWAQKAFL enterica enterica] ENDNCYVINNSDYDFSAIDMYLMSLCKNNIIANSTYSWWGAWLNKYEDKLVISPKQWFLGNNETSLRNAS subsp. WITL enterica serovar Poona str. ATCC BAA-1673 Bacteroides WP_ 547748823 glycosyl- 26.01 FutZA MRLIKMTGGLGNQMFIYAFYLRMKKRYPKVRIDLSDMNHYHVHHGYEMHRVFNLPHYEFCINQPLKKVIEF 21 sp. CAG:633 022161880.1 transferase LFFKKIYERKQDPNSLRAFEDDYLWPLLFKGFYQSERFFADIKDEVRKAFTFDSSVKVARSAELLRRLDADAN family 11 AVSLHIRRDGYLQPQHWATTGSVCQLPYYQNAIAEMNRRVAASPYYVFSDDIAWVKENIPLQNAVYIDWN [Bacteroides KGEESWQDMMLMSHCRHHIICNSTFSWWGAWLDPHEDKIVIVPNRWFQHCETPNIYPAGWVKVAIN sp. CAG:633] Clostridium WP_ 547839506 alpha-1-2- 34.28 MEKIKIVKLQGGMGNQMFQYAFGKGLESKFGCKVLFDKINYDELQKTIINNTGKNAEGICVRKYELGIFNLNI 22 sp. CAG:306 022247142.1 fucosyl- DFATAEQIQECIGEKLNKACYLPGFIRKIFNLSKNKTVSNRIFEKKYGEYDEEILKDYSLAYYDGYFQNPKYFEDI transferase SDKIKKEFTLPEIKNHDIYNKKLLEKITQFENSVFIHVRRDDYLNINCEIDLDYYQKAVKYILKHIENPKFFVCAE [Clostridum DPDYIKNHFDIGYDFELVGENNKTQDTYYENMRLMMACKHAIIANSSYSWWAAWLSDYDNKIVIAPTPWL sp. CAG:306] PGISNEIICKNWIQIKRGISNE Prevotella WP_ 497004957 protein 32.11 MKIVKILGGLGNQMFQYALYLSLQESFPKERVALDLSCFNGYHLHNGFELERIFSLTAQKASAATIMRIAYYYP 23 sp. oral 009434595.1 [Prevotella NYLLWRIGKRLLPRRKTMCLESSTFRYDESVLTREGNFRYFDGYWQDERYFVACREKVLKAFTFPAFKRTENLS taxon 306; sp. oral LLRKLDKNSVAIHVRRGDYIGNQLYQGICDLDYYRAAIDKISTYVTPSVFCIFSNDIAWCQTHLQPYLKAPVVY Prevotella taxon 306] VTWNTGTESYRDMQLMSCCAHNIIANSSFSWWGAWLNQNNEKVVIAKRWLNMDDCQFPLPASWVKI sp. oral  taxon 306 str. F0472 Brachyspira WP_ 547139308 glycosyl 30.14 MQLVKLMGGLGNQMFQYAFAKALGDKNILFYDGYKKHSLRKVELNRFKCKAVYIPRELFKYLKFVFTKFDKIE 24 sp. CAG:484 021917109.1 transferase YMRSDIYVPEYLNRDGNHIYIGFWQTEKYFKQIRPRLLKDFTPRKKLDRENAGIISKMQQINSVSVHIRRTDYV family 11 DESHIYDGTNLDYYKRAIEYSSKIENPEFFFFSDDMAYVKEKFAGLKFPHSFIDINSGNNSYKDLILMKNCKHN [Brachyspira IIANSTFSWWGAWLNENEEKIVIAPAKWFVTGENKDKIVPDEWIKL sp. CAG:484] Thalassospira WP_ 496164823 glycosyl 30. MVIVKLLGGLGNQMFQYATGRAVASRLDVELLLDVSAFAHYDLRRYELDDWNITARLATSEELARSGVTAA 25 profundimaris; 008889330.1 transferase PPSFFDRIAFLRIDLPVNCFREASFTYDPRILEVSSPVYLDGYWQSERYFLDIEKKLRQEFQLKASIDANNHSFK Thalassospira family 11 KKIDGLGKQAVSLHVRRGDYVTNPQTASYHGVCSLDYYRAAVDYIAEHVSDPCFFVFSDDLEWVQTNLNIK profundimaris [Thalassospira QPIVLVDANGPDNGAADMALMMACRHHIIANSSFSWWGSWLNPLNDKIIVAPKKWFGRANHDTTDLVP WP0211 profundimaris] DSWVRL Acetobacter WP_ 547459369 alpha-1 2- 29.99 MAVSPQESKYSAHVSPDKPLRIVRLGGGLGNQMFQYAFGLAAGDVLWDNTSFLTNHYRSFDLGLYNISGDF 26 sp. CAG:267 022078656.1 fucosyl- ASNEQIKKCKNEIRFKNILPRSIRKKFNLGKFIYLKTNRVCERQINRYEPELLSKDGDVYYDGVFQTEKYFKPLRE transferase RLLHDFTLTKPLDAANLDMLAKIRAADAVAVHIRRGDYLNPRSPFTYLDKDYFLNAMDYIGKRVDKPHFFIFS [Acetobacter SDTDWVRTNIQTAYPQTIVEINDEKHGYFDLELMRNCRHNIIANSTFSWWGAWLNTNPDKIVVAPKQWFR sp. CAG:267] PDAAEYSGDIVPNKDWIKL Dysgonomonas WP_ 493896281 protein 29.9 MVTVLLSGGLGNQMFQYAAAKSLAIRLNTALSVDLYTFSKKTQATVRPYELGIGNIEDVVETSSLKAKAVIKAR 27 mossii; 006842165.1 [Dysgonomonas PFIQRHRSFFQRFGVFTDTYAILYQPTFEALTFFVIMSGYFQNESYFKNISELLRKDFSFKYPLIGENKDVAGQI Dysgonomonas mossii] SENQSVAVHIRRGDYLNKNSQSNFAILEKDYYEKAINYISAHVKNPEFYVFSEDFDWIKDNLNFKEFPVTFID mossii DSM WNKGKDSYIDMQLMSLCKHNIIANSSFSWWSAWLNNSEERKIVAPERWFVDEQKNELLDCFYPQGWIKI 22836 Clostridium WP_ 545396671 glycosyl- 29.83 >gi|545396671|ref|WP_021636924.1|glycoyltransferase, family 11 [Clostridium 28 sp. KLE 021636924.1 transferase, sp. KLE 1755]MIIEISGGLGNQMFQYALGQKFISMGKEVKYDLSFYNDRVQTLRQFELDIFDLDCPVASNSELS 1755 family 11 GNSLKSRLKQKLGWDKEKIYEENLDLGYQPRIFELDDIYSGYWQDSELYFKDIREQILRLYTFPIQLDYMNGVFL [Clostridium RKIENSNSVSIHIRRGDYLNENNLKIYGNICTLNYYNKALQIIAKKITNPIIFVFTNDIEWVRKELEIPNMVIVDC sp. KLE 1755] NSGKLSYWDMYLMSKCKANIVANSSFSWWGAWLNKNENRIIISPKRWLNNHEQTSTLCDNWIRCGDD Gillisia WP_ 494045950 alpha-1,2- 29.28 MFISKNTVIIKLVGGLGNQMFQFAIAKIIAEKEKSEVLVDITFYTELTENTKKFPRHFSLGIFNSSFAIASKKEIDYF 29 limnaea; 006988068.1 fucosyl- TKLSNFNKFKKKLGLNYPTIFHESSFNFKAQVLELKAPIYLNGYFQSFRYFLGKEYVIRKIFKFPDEALDKDNDNI Gillisia transferase KRKIIGKTSVSLHIRRGDYVNNKKTQQFHGNCTIDYYQSAIAYLSSKLTDFNLIFFSDDIHWVRQQFKNISNQKI limnaea DSM [Gillisia YVSGNLNHNSWKDMYLMSLCDHNIIANSSFSWWGAWLNKNPEKIIIAPKRWFADTEQDKNSIDLIPSEWY 15749 limneas] RI Methylotenera YP_ 253996403 glycosyl 29.19 MLVSRIIGGLGNQMFEYAAARAASLRISVQLKLDLSGFETYDLHAYGLNNFNIVEDVAKKDDYFIGAPESLLKK 30 mobilis; 003048467.1 transferase IKKYLRGLIQLESFRESDLSFDSKVLELNDNTYLDGYWQCERYFIDFDKQIRQDFSFKFAPDALNQRYLELIDSV Methylotenera family protein NAVSVHIRRGDYVSNSTTNEIHGVCDLDYYQRAAEFMRARIGPENLHFFVFSDDTDWVKENISFGSDTTFIS mobilis JLW8 [Methylotenera HNDAAKNYEDMRLMSACKHHIIANSSFSWWAAWLNPSKQKVVIAPRQWFKSTLLNSDDIVPASWVRL mobilis JLW8] Runella YP_ 338214504 glycosyl 29.14 WMMVKLSGGLGNQLFQYAFGRHLATVNQKELKLDTSALTKTSDWTNRSYALDAFNIRAQEATPEEIKALAGKP 31 slithyformis; 004658567.1 transferase NRLLQRVGRKVGITPIQYFQEPHFHFYSSALSIKSSHYLEGYWQSEKYFEAITPILREEFAFTISPSTHAQTIKEKI Runella family protein SNGTSVSIHLRRGDYVKTSKANRYLRPLTMDYYQKAIDYINQRVKNPNFFLFSDDIKWAKSQVTFPPTTHFST slithyformis [Runella GTSAHEDLWLMTHCRHHIIANSTFSWWGAWLNQQPDKIVIAPQKWFSTERFDTKDLLPEPWIQL DSM 19594 slithyformis DSM 19594] Pseudo- WP_ 489048235 alpha-1,2- 29.1 MIKVKAIGGLGNQLFQYATARAIAEKRGDGNNNDMSDFSSYKTHPFCLNKFRCKATYESKPKLINKLLSNEIKR 32 alteromonas 002958454.1 fucosyl- NLLQKLGFIKKYFETQLPFNEDVLLNNSINYLTGYFQSEKYFLSIRECLLDELTLIEDLNIAETAVSKAIKNAKNSI haloplanktis; transferase SIHIRRGDYVSNEGANKTHGVCDSDYFKKALNYFSERKLLDEHTELFIFSDDIEWCRNNLSFDYKMNFVDGSS Pseudo- [Pseudo- ERPEVDMVLMSQCKHQVISNSTFSWWGAWLNKDEKVVVAPKEWFKSTDLDSTDIVPNQWIKL doalteromonas alteromonas haloplanktis haloplanktis] ANT/505 uncultured EKE06679.1 406985989 glycosyl 28.67 MLTLKLKGGLGNQMFQYAASHNLAKNKKTKINFDLSFFSDIEVRDIKRDYLLDKFNISADISFDQKNISGFRK 33 bacterium transferase FLVKVISKFFGEVYFYYRLKFLSSKYLDGYFQSEKYFKNVEEDIRKDFTLDKDEMGVEAKKIEQQIVNSKNSVLHIR family 11 RGDYVDDLKTNIYHGVCNLDYYKRSIKYLKENFGEINIFVFSDDIAWVKENLAFENLQFVSRPDIKDYEELML [uncultured MSKCEHNIIANSSFSWWGAWLNENKNKIIIAPKEWFQKFNINEKHIVPKSWIRL bacterium] Clostridium WP_ 545396696 glycolsyl- 28.57 MVIVQLSGGLGNQMFEYALYLSLKAKGKVVKIDDITCYEGPGTRPKQLDVFGVSYERATKQELTEMTDSSLD 34 sp. KLE 1755 021636949.1 transferase, PVSRIRRKLTGRKTKAYREKDINFDPQNMERDPALLEGCFQSEKYFQDCREQVREAYRRGIESGAYPLPEAY family 11 RRLEKEIADCKSVSVHIRRDGYLEESHGGLYTGICTEQYYQEAFARMEKEVPGAKFFLFSNDPDWTREHFKGE [Clostridium NRILVEGSTEDTGYLDLYLMSKCKHNIIANSSFSWWGAWLNDNPEKKVTAPWLNGRECRDIYTERMIRI sp. KLE 1755] Francisella WP_ 490414974 alpha-1,2- 28.57 MKIIKIQGGLGNQMFQYAFYKSLKNNCIDCYVDIKNYDTYKLHYGFELNRIFKNIDLSFARKYHKKEVLGKLFSI 35 philomiragia; 004287502.1 fucosyl- IPSKFIVKFNKNYILQKNFAFDKAYFEIDNCYLDGYWQSEKTFKKITKDIYDAFTFEPLDSINFEFLKNIQDYNLV Francisella transferase SIHVRRGDYVNHPLHGGICDLEYYNKAISFIRSKVANVHFLVFSNDILWCKDNLKLDRVTYIDHNRWMDSYK philomiragia [Francisella DMHLMSLCKHNIIANSSFSWWGAWLNQNDDKIVIAPSKWFNDDKNIQKDICPSNWVRI subsp. philomiragia] philomiragia ATCC 25015 Pseudomonas WP_ 515906733 protein 28.52 MVIAHLIGGLGNQMFQYAAARALSSAKKEPLLLDTSSFESYTLHQGFELSKLFAGEMCIARDKDINHVLSWQ 36 fluorescens; 017337316.1 [Pseudomonas AFPRIRNFLHRPKLAFLRKASLIIEPSFHYWNGIQKAPADCYLMGYWQSERYFQDAAEEIRKDFTFKLNMSPQ Pseudomonas fluorescens] NIATADQILNTNAISLHVRRGDYVNNSVYAACTVEYYQAAIQLLSKRVDAPTFFVFSDDIDWVKNNLNIGFPH fluorescens CYVNHNKGSESYNDMRLMSMCQHNIIANSSFSWWGAWLNSNADKIVVAPKQWFINNTNVNDLFPPAW NCIMB 11764 VTL Herbas- WP_ 495392680 glycosyl 28.48 MIATRLIGGLGNQMFQYAAGRALALRVGSPLLLDVSGFANYELRRYELDGFRIDATAASAQQLARLGVNATP 37 pirillum sp. 008117381.1 transferase GTSLLARVLRKVWPAPADRILREASFTYDARIEQASAPVYLDGYWQSERYFARIRQHLLDEFTLKGDWGSDN Yr522 family 11 AAMAAQIATAGAGAVSLHVRRGDYSNAHTAQYHGVCSLDYYRDAVAHIGGRVEAPHFFVFSDDHEWVR [Herbase- ENLQIGHPATFVQINSADGIYDMMLMKSCRHHIIANSSFSWWGAWLNGPAEDKIVVAPQRWFKDATNDT pirillum sp. RDLIPAAWVRL YR522] Prevotella WP_ 496097659 protein 28.43 MKIVKILGGLGNQMFQYALYLSLKETFPQENVTVDLSCFHGYHLHNGFEIARIFSLHPDKATVMEILRIAYYYP 38 histicola; 008822166.1 [Prevotella NYFFWQIGKRVLPQRKTMCTESTKLLFDKSVLQREGDRYDGYWQDERYFIDCRRTILNTFKFPPFTDDNNL Prevotella histicola] ALLKKMDTNSVSIHVRRGDYMGNKLYQGICKLNYYREAIMKISSYISPSMFCVFSNDIEWCRDNLESFIKAPIY histicola YVDWNSGTESYRDMQLMSCCGHNIIANSSFSWWGAWLNQNSSKIVIAPKRWINLKNCGFMLPSRWVKI F0411 Flavo- WP_ 516064371 protein 28.42 MIVVQLIGGLGNQLFQYAAAKALALQTKQKFSLDVSQFESYKLHNYALNHFNVISKNYKKPNRYLRKIKSFYQ 39 bacterium sp. 017494954.1 [Flavo- KNVFYKEVDFGYNPDLIHLKGGIIFLEGYFQSEKYFIKYEKEIREDFELRTPLKKETKAAIAKIESVNSVSIHIRRGD WG21 bacterium YINNPLHNTSKEEYYNKALEIVENKINNPVYFVSDDMEWVKANFSTKQETIFIDFNDASTNFEDLKLMTSCK sp. WG21] HNIIANSSFSWWGGWLNKNPDKIVIAPKRWFNDDSINTNDIIPTNWKVI Polaribacter WP_ 517774309 protein 28.42 MIIVRIVGGLGNQMFQYAYAKALQQKGYQVKIDITKFKKYNLHGGYQLDQFKIDLETSSPIANVLCRIGLRRS 40 franzmannii 018944517.1 [Polarbacter VKEKSLLFDEKFLEIPQREYIKGYFQTEKYFSSITPILRKQFIVQKELCNTTLRYLKEITIQKNACSLHIRRGDY franzmannii] ISDEKANSVHGTCDLPYYKKSIKRIQFYKDAHFFIFSDDISWAKKNLLITNATYIDHNVIPHEDMYLMTLCHNI ANSSFSWWGAWLNQHENKTVIAPKNWFVNRENEVACANWIQL Polaribacter WP_ 472321325 glycosyl 28.42 MVVVRILGGLGNQMFQYAYAKSLAEKGYEVQIDISKFKSYKLHGGYHLDKFRIDLETANSSSAFLSKIGLKKTIK 41 sp. MED152 07670847.1 transferase EPNLLFHKDLLKVNNNAFIKGYFQAEQYFSDIREILINQFKIKKELAKSTLAIKNQIELLKTTCSHLVRRDGYISDK family 11 KANKVHGTCDLDYYSSAIEDHISKQNSNVHFFVFSDDIAWVKDNLNITNARYDHNVIPHEDMYLMTLCNHNI [Polarbacter TANSSFSWWGAWLNQNPDKIVIPKNWFVDKENEVACKSWITL sp. MED152] Mehtano- YP_ 150402264 glycosyl 28.19 MKIIQLKGGLGNQMFQYALYKSLKKRGQEVLLDISWYLKNNAHNGYELEWVFGLSPEYASIRQCFKLGDIPI 42 coccus 001329558.1 transferase NLIYNVKRKVFPKKTHFFEKSNFNYDNNVFEVTNGYFEGWQNENYFKNFRSEILNDFSFKNIDKRNAEFSEY maripaludis; family protein LKSINSVSVHVRRGDYVTNQKALNVHGNICNLEYYNKAINLANNNLKNPKFVIFSDDITWCKSNLGIDDPVYV Methano- [Methano- DWNTGPYSYQDMYLMSNCKNNIIANSSFSWWGAWLNQNTEKKVFSPKKWVNDRNNVNIVPNGWIKIK coccus  coccus maripaludis maripaludis C7 C7] Gallionella WP_ 517104561 protein 28.15 MIIAHIIGGLGNQMFQYAAGRALSLARGVPFKLDISGFEGYDLHQGFELQRVFNCAAGIASEAEVRDSLGW 43 sp. SCGC 018293379.1 [Gallionella QFSSPIRRIVARPSLAVLRRSTFVVEPHFHYWAGIKQVPDNCYLAGYWQSEQFQSHAAVIRTDFAFKPPLSG AAA018-N21 sp. SCGC QNSKLAMQIAQGNAVSLHIRRGDYANNPKTTATHGLCSLDYYRAAIQHIAERVQSPHFFIFSDDIAWVKSNL AAA018-N21] AINFPHQVYGHNQGTESYNDMRLMSLCQHNIIANSSFSWWGAWLNTNAHKIVIAPKQWFANTTHVADLI Azospira YP_ 372486759 Glycosyl 28.04 MQSPACIAGARAWWVGYGMAEAMQPVVVGLSGGLGNQMFQYAAGRALAHRLGHPLSLDLSWFQGRG 44 oryzae; 005026324.1 transferase DRHFALAPFHIAASLERAWPRLPPAMQAQLSRLSRRWAPRIMGAPVFREPHFGYVPAGAALAAPVFLEGY Dechlorosoma family 11 WQSERYFRELREPLLQDFSLRQPPLPASCQPILAAIGNSDAICVHVRRGDYLSNPVAAKVHGVCPVDYYQQGV suillum PS [Dechlorosoma AELSASLARPHCFVFSDDPEWVRGSLAFPCPMTVVDVNGPAEAHFDLALMAACQHFVIANSSLSWWGAW suillum PS] LGQAAGKRVIAPSRWFLTSDKDARDLLPPSWERR Prevotella WP_ 517274199 protein 28 MKIVKIIGGLGNQMFQYALAMALNKNFTKEEVKLDIHCFNGYTKHQGFEIDRVFGNEFELASYRDVAKVAY 45 paludivivens 018463017.1 [Prevotella PYFNFQLWRIGSRIFPDRRHMISEDTSFKIMPEVITSHNYKYYDGYWQHEEYFKNIHDEILDAFKPKFQDER paludivivens] NKALAERLSDSNSISIHIRRGDYLNDELFKGTCGIEYYKKAIEEINERTVPTLFCVFSNDIHWCKENIEPLLNGKE TIYVDWNTGSDNYRDMQLMTKCKHNIIANSSFSWWGAWLNNTKDKIVIAPRIWYNTKEKVSPVASSWIKL Gramella YP_ 120434923 alpha-1,2- 27.96 MSNKNPVIVEIMGGLGNQMFQFAVAKLLAEKNSSVLLVDTNFYKEISQNLKDFPRYFSLGIFDISYKMGTEN 46 forsetii; 860609.1 fucosyl- GMVNFKNSLFKNRVSRKLGLNYPKIFKEKSYRFADLFNKKTPIYLKGYFQSYKFIGVESKIRQWFEFPYENL Gramella transferase GVGNEEIKSKILEKTSVSVHIRRGDYVENKKTKEFHGNCSLEYYKNAITYFLDIVKEFNIVFFSDDISWVRDEFK forsetii  [Gramella DLPNEKVFVTGNLHENSWKDMYLMSLCDHNIIANSSFSWWAAWLNNNSEKNVIAPKKWFADIDQEQKSL KT0803 forsetii DLLPPSWIRM KT0803] Mariprofundus WP_ 497634831 alpha-1,2- 27.92 MIIVQFTGGLGNQMFQYALGRRLSLLHDVELKFDLSFYQHDILRDFMLDRFQVNGQVATEKEIEAYTNTPIF 47 ferrooxyclans; 009849029.1 fucosyl- ALDRPLLDRLVRWGLYRGIVSVSDEPPGKQALMNYNSRVLQAPRNTYVQGYWQSEKYFMPIRQKLLDDFSL Mariprofundus transferase VDKADQANGAMLEKIRQCHSVSLHVRRGDYVSNPLTNHSHGTCGLEYYEKAIALIGSKVDDPHFFVFSDDPE ferrooxydans [Mariprofundus WTRDHLKCRFPMTVTCNSADSCEWDMELMRHCRDHIIANSSFSWWGAWLNMNPDKVVVAPAAWFN PV-1 ferrooxydans] NFSADTSDLIPDSWVRI Bacillus WP_ 488102896 protein 27.91 MKIIQVSSGLGNQMFQYALYKKISLNDNDVFLDSSTSYMMYKNQHNGYELERIFHIKPRHAGKEIIKNLSDLD 48 cereus; 002174293.1 [Bacillus SELISRIRRKLFGAKKSMYVELKEFEYDPIIFEKKETYFKGYWQNYNYFKDIEQELRKDFVFTEKLDKRNEKLANE Bacillus cereus] IRNKNSVSIHIRRGDYYLNKYEEKFGNIANLEYYLKAINLVKKKIEDPKFYFSDDIDWAQKNINLTNDVVYISH cereus VD107 NQGNESYKDMQLMSLCKHNIIANSTFSWWGAFLNNNDDKIVVAPKKWINIKGLEKVELFPENWITY Firmicutes WP_ 547951299 protein 27.81 MIIIRMTGGLGNQMFQYALYLKLRAMGKEVKMDDFTEYEGREARPLSLWAFGIEYDRASREELCRMTDGFL 49 bacterium 022352106.1 [Firmicutes DPVSRIRRKLFGRKSLEYMEKDCNFDPEILNRDPAYLTGYFQSEKYFADIEEEVRQAFRFSERIWEGIPSQLLER CAG:534 bacterium IRSYEQQIKTTMAVSVHIRRGDYLQNEEAYGGICTERYYKTAIEYVKKRQQDASFFVFTNDPDYAGEWILKNF CAG:534] GQEKERFVLIEFTQEENGYLDLYLMSLCRHHILANSSFSWWGAYLNPSREKMVIVPHKWFGNQECRDIYME NMIRIAKEQS Sideroxydans YP_ 291615344 glycosyl 27.81 MVISNIIGGLGNQMFQYAAARALSLKLEVPLKLDISGFTNYALHQGFELDFRIGCKIEIASEADVHEILGWQSA 50 litho- 003525501.1 transferase SGIRRVVSRPGMSIFRRKGFVVEPHFSYWNGIRKITGDCYLAGYWQSEKYFLDAAVEIRKDFSFKLPLDSHNA trophicus; family 11 ELAEKIDQENAVSLHIRRGDYANNPLTAATHGLCSLDYYRKSIKHIAGQVRNPYFFVFSDDIAWVKDNLEIEFP Sideroxydans [Sideroxydans SQYVDYNHGSMSFNDMRLMSLCKHHIIANSSFSWWGAWLNPNPEKVVIAPERWFANRTDVQDLLPPGW litho- litho- VKL trophicus; trophicus  ES-1 ES-1] zeta WP_ 517092760 protein 27.81 MIVSQIIGGLGNQMFQYATGRALSHRLHDTFFLDLDGFSGYQLHQGFELSNVFQCEVNVATRSQMQALLG 51 proteo- 018281578.1 [zeta WRSFSSVRRLLMKRSLKWARGHRVMIEPHFHYWSRFAEINEGCYLSGYWQSERYFKPIENIIRQDFKFNHLL bacterium proteo- KGVNLDLAQQMETVNSNSLHVRRGDYASDANTNHTHGLCPLDYYRDAILYIAQNTVAPSFFIFSDDIEWCRE SCGC AB-137- bacterium  HLKLSFPATYIDHNKGSNSYCDMQLMSLCHHHIIANSSFSWWGAWLNTRLDKVIAPKQWFANGNRTDDLI C09 SCGC AB-137- PAEWLVM C09] Pedobacter YP_ 255530062 glycosyl 27.8 MKIIRFLGGLGNQMFQYAFYKSLQHRFPHVKADLQGYQEYTLHNGFELEHIFNIKVNSVSSFTSDLFYNKKW 52 heparinus; 003090434.1 transferase LYRKLRRILNKLRNTYIEEKKLFSFDPSLLNNPKSAYYWGYWQNFQYFEHIADDLRKDFQFRAPLSAQNQEVLD Pedobacter family protein QTKLSNSISLHIRRGDYIKDPLLGGLCGPEYYQTAINYITSKVNAARFFIFSDDIDWCIANLKLQDCSFISWNKG heparinus; [Pedobacter TSSYIDMQLMSSCKHHIVANSSFSWWAWLNPNPDKIVIAPEKWTNDKDINVRMSFPQGWISL DSM 2366 heparinus DSM 2366] Methylophilus WP_ 517814852 protein 27.78 MFQYAMGLSLAENNQTPLKLDLSQFTDYKLHNGFELSKVFNCSAETASVTQIETLLGICKYSFIRRILKNTYLKN 53 methylotrophus 018985060.1 [Methyl- LRPAQYVVEPFFGYWDGVNFLGDNVYLEGYWQSQKYFIDYESTIRTHFTFKNILSGENLKLSDRIKGSNSVSL ophilus HIRRGDYVTNKNNAFIGTCSLIYYQNAIEYFSTKIADPIFFIFSDDITWAKSNLRLANEHYFVGHNQGEDSHFD methylo- MQLMSLCKHHIIANSSFSWWGAWLNPSKDKIIIAPKKWFASGLNDQDLVPKDWLRI trophus] Rhodo- WP_ 495309205 alpha-1,2- 27.7 MIYTRIRGGLGNQLFQYSAARSLADYLNVSLGLDTREFDENSPYKMSLNHGNIRADLNPPDLIKHKKDGKIAYI 54 bacterales 008033953.1 fucosyl- IDHIKGNQKKVYKEPFLSFDKNLFSNVDGTYLKGYWQSEKYFLRNRKNILSDINLIKKTDKFNTINLKEIKKSTSI bacterium transferase  SLHIRRGDYLSNESYNETHGICSLSYYTDAVEYIKNRLGENIKVFAFSDDPDWVLENLKLSVDIKIINNNTSANS HTCC2255 [Rhodo- FEDLRLMLNCDHNIIANSSFSWWGAWLNQNPEKIVISPKKWYNKKQLQNADIVPSSWLKV bacterales bacterium HTCC2255] Spirulina WP_ 515872075 protein 27.69 MAKIIARIGGIGNQIFIYAAARRLELINNAELVLDSVSGFVHDLQYRQHYQLDHFHIPCRKATPAERFEPFSR 55 subsalsa 017302658.1 [Spirulina VRRYLKRQLNQRLPFEQRRYVIQESIDFDPRLIEFKPRGTVHLEGYWQSEDFYKDIEATIRQDLQIQPPTDPTN subsalsa] LAIVQHIHQHTSVAVHIRFFDQPNADTMNNAPSDYYHRAVEAMETFVPGAHYYLFSDQPEAAKSRIPLPDE RVTLVNHNRGNKLAYADLWLMTQCQHFIIANSTFSWWGAWLAENQKKQVIAPGFEKREGVSWWGFKGL LPKQWIKL Vibrio WP_ 498119755 glycosyl 27.67 MVIVKITGGLGNQLFQYATGSALANKLSCELVLDLSFYPTQTLRKYELAKFNINARVATDREIFLAGGGNDFFS 56 cyclitrophicus 010433911.1 transferase KALKKLGLTSIIFPEYIKEQESIKYVGKIDLCKSGAYLDGYWQNPLYFSQNKIELTREFLPRAQLSPASALAWKDHI family 11 SQASNSVSLHVRRGDYVENAHTNNIHGTCSLEYYQHAIEKIRSEVHNPVFFVFSDDIEWCKLNLSSLAEVEFV [Vibrio DNTTSAIDDLMLMRQCKHSIIANSTFSWWGAWLKLDGLVIAPRNWFSSASRNLKGIYPKEWHIL cyclitophicus] Lachno- WP_ 551039510 protein 27.65 MRSVVDIKGGYGNQLFCYSFGYAVSKETGSELIIDTSMILDMNNVKDRNYQLGVLGITYDSHISYKYGKDFLSR 57 spiraceae 022783177.1 [Lachno- KTGLNRLRKKSAIGFGTVVFKEKEQYVYDPSVFEIKRDTYFDGFWQSSRYFEKYSDDLRKMLKPKKISNAAEKL bacterium spiraceae AEDARDCLSVSVHIRRGDYVSLGTWLKDDYYIKALDIIKERYGSEPVFFVFSDNKKYADDFFSAAGLKYRLMD NK4A179 bacterium YETDDAVRDDMFLMSRCSHNIMANSSYSWWGAFLNDNKDKTVICPETGVWGGDFYPEGWMKVTASSG NK4A179] K uncultured EKE02186.1 406980610 glycosyl 27.57 MIIVNLYGGLGNQMFQYALGRHLAEKNNTELKLDISAFESYKLRKYELGNLNIIEKFALPEEISRLSTLPTGKIER 58 bacterium transferase FIRKTLRKPVKKPESYKENITGGFNPKILDLQNNIYLEGYWQSEKYFIEIEDIIRKEPSFKPATGKKNEILENILNI family protein NSVSLHIRRGDYVTNPEVNQNHGVCSLDYYKSCVDFIEKKLESPYFYIFSDDIEWVKNNLQIQSQVYYVDHNT [uncultured VDNAIEDMRLMFSCHNKILANSSFSWWGAWLNSNPDKMVITPRKWFNTTYDSNDLIPERWIKL bacterium] Bacteroides WP_ 492366053 protein 27.46 MKIGIIYIVTGPYIKRWNEFYSSSQLYFCVEAEKNYEVFTDSSELASQRLPNVHMHLIEDKGWIVNVSSKSVFIC 59 fragilis; 05822375.1 [Bacteroides EIRNQLTSYDYIFYLNGNFKFISPIYCDEILPQAEHNYLTALSFSHYLTIHPDHYPYDRNKNCNAFIFYGQGKYYF Bacteroides fragilis] QGGFYGGRTQEVLSLSEWCRDAIEADFNKKVIARFHDESYINRYLLTQHPKVLNKKYAFQDIWPYEGEYKAIV fragilis  LNKEEVPEDNNLQEMKQNYIDPSLSFLLNDELKFIPISIVQLYGGLGNQMFGYAFYLYIRHISTQERKLLIDPAP HMW 616 CKRYGNHNGYELPSIFSKICQHIHISDETKNNIRKLRKGTSLSIEEVRASMPQSFKEKKQPIIFYSGCWQCVTYV ETVKDEIKKDFIFDESKLNEPSAQMLRIIRRSNSVNVHIRRNDYLIGNNEFLYGGICTKSYYEKAISQMYTLLKDE PIFIYFTDDPEWVRSNFALDKSYLVDWNKNKDNWQDMYLMSACRHHIIANSSFSWWAAWLGGFPEKKVI APSTWLNGMQTPDILPTEWIKIPITPDKKILDRICNHLILHSSYMKQLGLNSGKMGVVIFFFHYARYTQNPLYE NYAGDLFDELYEEIHKGISFSFLDGLCGIAWAVEYLVHEQFIEGNTDDSLAEIDFKVMQIDPRRFTDYSFETGL EGIACYVLSRLLSPRVCSSSLTLDSVYLKDTLEACRKVPVDKANYTRLFLNYIESKEVGYSFKDVLMQVLNHSEK AFGSDGLTWQTGLTMIMR Butyrivibrio WP_ 551034739 protein 27.46 MIIIQLKGGLGNQMFQYALYKELRSRGKEVKIDDVTGFVDDELRTPVLQRFGIEYDRATREEVVKLTDSKMDI 60 sp. 022778576.1 [Butyrivibrio FSRIRRKLTGRKTCRIDEESGTFNPDILELDEAYLVGYWQSDKYFRNEDVIAQLRQEFQKRPQEIMTDSASWA AE3009 sp. TLQQIECCQSVSLHIRRTDYIDEEHNHIHNLCTEKYYKGAIDRIRSQYPSAVFFIFTDDKEWCRNHFRGPNFFV AE3009] VELAEKENTDIAEMLLMSSCKHHICANSSFSWWSAWLNDSPEKMVIVPNKWINNRDMDDIYTDRMTKM AI Bacteroides WP_ 490447027 protein 27.43 MKQTIILSGGLGNQMFQYAFFLSMKAKGKSCSLDTTLFQTNKMHNGFELKSVFDIPDSPNQASALHSLLIKM 61 avatus; 004317929.1 [Bacteroides LRRYKPKSILTIDEPYTFCPDALESKKSFLMGDWLSPKYFESIKDVVVNAYRFHNIGNKNVDTANEMHGNNS Bacteroides ovatus] VSIHIRRGDYLKLPYYCVCNENYYRQAEIEQIKDRVDNPIFYVFSNEPSWCDSFMKEFRVNFKIVNWNQGKDS ovatus YQDMYLMTQCKHNIIANSTFSWWGAWLNNNTDKIIVAPSKWFKNSEHNINCKEWLLIDTSK CL02T12C04 Desulfospira WP_ 550911345 protein 27.42 MGKKYVETVVNGGLGNQIFQFSAGFALSKRLNLDLVLNISTFDSCQKRNFELYTFPKIKNSFACIKDDDPGVFS 62 joergensenii 022664368.1 [Desulfospira RLRIPFLNFKEKIKQFHESHFFFDPAFFDIREPVRIEGYFQSYKYFEKYSDQLKDILLDIPLTSRLKTVLKVISSKES joergensenii] VSVHIRRGDYISDQGINEVHGTLNEAYYLNSIKLMEKMFPESFFFLFTDDPHYVEENFKFLEDTSCIISDNDCLP YEDMYLMANCHHNIIANSSFSWWGAWLNQNPEKIVIAPRKWFSRKILMEKPVMDLLPDDWILL Lachno- EOS74299.1 507817890 protein 27.39 MNIIRMTGGLGNQMFQYALFLRLKAQGKEVKFDDRTEYKGEEARPILLWAFGIDYPAAGEEEVNELTDGV 63 spiraceae C819_03052 MKFSHRLRRKLFGRKSKEYREKSCNFDQQILEKEPAYFTGYFQSERYFEEVKEQVRKAFQFSGKIWGSVSKEL bacterium [Lachno- EERIREYQTKIENKSQMPVSVHIRRGDYLENDEAYGGICTDAYYRKAIEMMEEKFPNTVFYIFSNDTGWAKQ 10-1 spiraceae WIDHFYKEKSRFIVIEGTTEDTGYLDLFLMSKCRAHIIANSSFSWWGAWLDPDQEKIVIAPSKWVNNQDMK bacterium DIYTREMIKISPKGEVR 10-1] Bacteroides WP_ 495107639 protein 27.33 MVVVYVGAGLARNMFQYAFALSLREKGLDVFIDEDSFIPRFDFERTKLDSVFVNVNIQRCDKNSFPLVLRED 64 dorei; 007832461.1 [Bacteroides RFYKLLKRISEYMSDNRYIERWNLDYLPIHKKASTNCIFIGFWISYKYFQSSEDAVRKAFTFKPLDSIRNVELAT Baceroides dorei] KLVTENSAVHFRKNIDYLKNLPNTCPPSYYYEAINYIKKVVPNPKFYFFSDNWDWVRENIRGVEFTAVDWN dorei PSSGIHSHCDMQLMSLCKHNIIANSTYSWWSAYLNENNNKIVVCPKDWYGGMVKKLDTIIPESWIING DSM 17855 Firmicutes WP_ 547127421 protein 27.32 MVKVKMSGGLGNQMFQYALYRKIQQTGKDVKLDLFSFQDKNAFRRFSLDIFPIEYQTANLEECRKLGECSYR 65 bacterium 021916201.1 [Firmicutes PVDKIRRKMFGLKESYYQEDLDKGYQPEILEMNPVYLDGYWQCERYFQDIREKILEDYTFPKKISIESSRLQERI CAG:24 bacterium KNTESVSIHIRRGDYLDAANYKIYGNICTIEYYQSAISRMRKLCEKPNFYLFSNDPEWAKEIFGDTEDTIVEEDK CAG:24] ERPDYEDMFLMSRCKHNIIANSSFSWWAAWLNQNENKRVIAPVKWFNNHSVTDVICDDWIRIDGDHKGA Clostridium WP_ 547299420 epsH 27.3 MIYVNIRGRLGNQLFIYAFARALQKSTNQQITLNYTSFRKHYNNTAMDLEQFNIPEDIMFENSKELPWFANT 66 hathewayi 022031822.1 [Clostridium DGKVIRILRHYFPKLIRSILQKMNVLMWLGDEYVEVKVNKRRDIYIDGFWQSSRYFKSVYKELKNELIPKMEM CAG:224 hathewayi SKEIKTMGDLINQKESVCVSVRRGDYVTVKKNRDYYICDEKYLNTSIMRMVELPNVTWFIFSDDADWVK CAG:224] DNIVFPGEVFYQPPRVTPLETLYLMKACKHFIISNSSFSWWGQYLSNNDNKIVIGPAKWYVDGRKTDIIEEE WIKIEV Syntrophus YP_ 85860461 alpha-1,2- 27.3 MVIVRLTGGIGNQMFQYAAARRVSLVNNAPLFLDLGWFQETGSWTPRKYELDAFRIAGESASVGDIKDFKS 67 acidi- 462663.1 fucosyl- RRQNAFFRRLPLFLKKRIFHTRQTHIIEKSYNFDPEILNLQGNVYLDGYWQSEKYFSDVDSEIRREFSFQTDPAE trophicus transferase RNRKILERIASCESVSIHIRRDGYVTLPDANAFHGLCTPAYYRLAVEQISRKVVEPVFFVFSDDIAWARGNLKL SB: [Syntrophus GFETCFMDQNGPDRGDEDLRLMIACRHHIIANSSFSWWGAWLCSNPEKIVYAPRKWFNNGLDTPDNIPAS Syntrophus acidi- WIRI acidi- trophicus trophus SB] Bacteriodes WP_ 491931393 protein 27.27 MKIVKIIGLGNQMFQYALYSLKKKYPKEKIKIDISMFETYGLHNGFELKRIFDIDAEYASREEIRELSFYIKIYKL 68 caccae; 005678148.1 [Bacteroides QRIFRKIFPVRKTECVEKYDFKFMSEVWSNCDRYYEGYWQNWEYFIEAQTEVRSTFTFKKELVGRNAKVIREI Bacteroides caccae] QYAKMPVSLHIRRGDYLHHKLFGGLCDLNYYKKAIDYVLNNYDTPQFVLFSNDIEWCKTYILPLVQGPFILVD caccae WNSGVESYIDMQLMSCCRINIIANSSFSWWAAWLNDSSEKIVIAPKLWAHSPYGKEIQLKSWLLF ATCC 43185 Butyrivibrio WP_ 551011888 protein 27.24 MIIIEMSGGLGNQMFQYALYKSMLHKGLDVTIDKSIYRDVDHKEQVDLDRFPNVSYIEADRKLSSTLRGYGY 69 fibrisolvens 022756304.1 [Butyrivibrio NDSIIDKIRNKLNKSKRNLYHEDLDKGYQPEIFEFDNYLNGYWQCERYFKDIKNEIKKDFIFPCTQSGDDKIK fibrisolvens] ALTIEMESCNSVSLHVRRGDYLKPGLIEIYGNICTEEYYKKSIEYIKERVDNPVFYIFSNDMAWVRDNFKSDDFR YVNEDGAFDGMTDMYLMTRCRHHIVANSSFSWWGAWLNKHDDNIVICPNRWVNTHTVTDIICEDWIRI DV Para- WP_ 492476819 protein 27.24 MIVGGNDYCKVKVVNIIGGLGNQMFQYAFALSLKEHFPKEEIRIDISHFNYLFVNKVGAANLHNGYELDKIFF 70 bacteroides 005857874.1 [Para- NIELKKANAWQLMKLTWFIPNYLISRIARKILPVRNSEYIQNSSDCFFYDPMVYNKQGSCYYEGYWQAIGYYE distasonis; bacteroides SMRDKLCKIFQHPSPEGKNKQYIENMESSNSVGIHIRRGDYLLSDNFRGICEVDYYKRAIDKILQDGEKHVFYL Para- distasonis] FSNDQKWCEEYILPLLGNYEIIFVTGNIGRDSCWDMFLMTHCKDLIIANSSFSWWGAFLNKRGGRVVTPKR bacteroides WMNRNIRYDLWMPEWIRI distasonis CL03T12C09 Geobacter YP_ 148263741 glycosyl 27.21 MIIARLQGGLGNQMFQYAVGLHLALTHNVELKIDITMFSDYKWHTYSLRPFNIRESIATEEEIKALTDVKMDR 71 uranii- 001230447.1 transferase PYKKIDNFLCRLLRKSQKISATHVKEKHFHYDPDILKLPDNVYLDGYWQSEKYFKEIENIIRQTFIIKNPQLGRDK reducens; family protein ELACKILSTESVCLHIRRGNYVTDKTTNSVLGPCDLSYYSNCIKSLAGNNKDPHFFVFSNDHEWVSKNLKLDYP Geobacter [Geobacter TIYVDHNNEDKDYEDLRLMSQCKHHIIANSTFSWWSAWLCSNPDKVIYAPQKWFRVDEYNTKDLLPSNWLI uranii- uranii- L reducens Rf4 reducens Rf4] Lachno- WP_ 511026085 protein 27.21 MIIVKIYEGLGNQLFQYAFARSIQVNGKKVFLDTSGYTDQLFPLCRTSTRRRYQLNCFNIRIKEVEKKNIEKYSFL 72 spiraceae 01280341.1 [Lachno- IQEDMFGKLISKLAKLHLWMYKVTIQQNAQEYKESYLNTRGNVYYKGWFQNPKYFSSIRRLLLKEITPKYKIRI bacterium spiraceae PAELRELLQEDNIVAVHCRRGDYQYIRNCLPVNYYKKAMAYMKEKKLGVPRYLFFSDDLSWVKRQFGNKDN A4 bacterium A4] Colwellia YP_ 71282201 alpha-1,2- 27.15 MKVVRVCGGFGNQLFQYAFYLAVKHKFNETTKLDIHDMASYELHNGYELERIGNLNENYCSAEEKLAVQSTK 73 psychrery- 270849.1 fucosyl- NIFTKLLKEIKKYTPFIPRTYIKEKKHLHFSYQEVDLGTKDTSIYYRGSWQNPQYFNSIASEIREKLTFPEFTEPKSL thraea; transferase ALHQEISEHETVAVHIRRGDYLKHKALGGICDLPYYQNAIKEIEGLVEKPLFVIFSDDITWCRANINVEKVRFVD Colwellia [Colwellia WNSGEQSFQDMHLMSLCTHNIIANSSFSWWGAWLNANPNKIVISPNKWIHYTDSMGIVPSEWIKVETSI psychrery- psychrery- thraea 34H thraea 34H] Roseobacter WP_ 497495952 alpha-1,2- 26.96 MITSRLHGRLGNQMFQYAAARALAHRLGCGVALDGRGAELRGEGVLTRVFDLPLSAAPKLPPLKQHAPLRY 74 sp. MED193 009810150.1 fucosyl- GLWRGLGLAPRFRRERGLGYNTAFETWEDGCYLHGYWQSERYFEEISDLIRADFTFPDFSNRQNAEMAARI transferase MEDNAISLHVRRGDYVALSAHVLCDQAYYEAALTRLLEGLSQDAPTVYVFSDDPDWAKANLPLPCKKVVVD [Roseobacter FNGPETDFEDMRLMSLCKHNIIGNSSFSWWAAWLNANPQKRVAGPANWFGDPKLSNPDILPSQWLKVA sp. MED193] P Cesiribacter WP_ 496488826 Glycosyl 26.89 MMIVRLCGGLGNQLFQYAVGKQLSVKNNIPLKIDDSWLRLPDARKYRLQFFQIEEPLASPQEVERFVGPYES 75 andamanensis; 009197396.1 transferase QSLYARLYRKVQNMLPRHRRRYFQESGFWAYEPELMRIRSQVFLEGFWQHHAYFTRLHPQVLEALQLREEY Cesiribacter family 11 RQEPYAVLDQIREDAASVSLHIRRGDYVSDPYNLQFFGVMPLSYYQQVAYMQEQLHAPTFYIFSDDLDWA andamanensis [Cesiribacter RAHLKLQAPMVFVDIEGGRKEYLELEAMRLCRHNILANSSFSWWGAYLNTNPHKRVIAPRQWVADPELKD AMV16 andamanensis] KVQIQMPDWILL Rhodo- WP_ 495954476 glycosyl 26.89 MIATRLIGGLGNQMFQYAYGFSLARRRSERLVLDVSAFESYDLHALAIDQFDISAARMTQAEFARIPGRYRG 76 pirellula 008679055.1 transferase KSRWAERVANFAGGLQSCDKRPLRLRREKPFGFAEKYLAEGSDLYLDGYWQSERYFPGLQAELKKEFQLKRG sallentina; family 11 LSDESSRVLDEIQSSMSVAMHVRRGDYVTNAETLRIYRRLDAEYYRKCLNDLRQRFSNLNVFVFSNDIQWCQ Rhodo- [Rhodo- DHLDVGLKQRPVTHNDATTAIEDMFLMSQCDHSIIANSSFSWWAAFLGRSDAQRRVYYPDPWFNPGTLN pirellula pirellula GDSLGCANWVSESSISVSRPSRAA sallentina; sallentina] SM41 Butyrivibrio WP_ 551018054 protein 26.85 MIIIRMMGGLGNQMFQYALYLQLKALGKEVKIDDVYGFRDDPQRDPVLEKMYGITYTKASDAEVVDITDSH 77 sp. AD3002 022762282.1 [Butyrivbrio LDIFSRIRRKLFGRKSHEYIEETGLFDPKVFEFETAYLNGYFQSDKYFPDKEVLAQLRREFVIKPDDVFTSADSE sp. AD3002] ELYRQIRETESVSIHVRRGDYLLPGTVETFGGICDNDYYKRAIDRMVSEHPDAIFFVFTSDKEWCEQNVSGKK FRIVDTKEENDDAADLLLMSLCKHHILANSSYSWWSAWMNDSPEKTVIVPSKWLNTKPMDDIYTSRMTKI Segetibacter WP_ 517440157 protein 26.78 MVVVKLIGGMGNQMFQYAIGRHLAIKNKCPLYFDHIELENKNTANTPRNYELDIFNVQYQKNPFLQSNRFV 78 koreensis 018611017.1 [Segetibacter AKVYHKLFSVQRIKEPDFTFHPHILNVQGNIHLNGYWQNENFKEIEEIIRQDFTFKTPANEKIESILQQIAATN koreensis] SVSLHVRRGDYITLTEANQFHGVCSDTYYQKAIAKIKEAIPAPHLFVFSDDIHWVKQNMPFTEEHTFVDGNT GKNSFEDLRLMAACRHNILANSSFSWWAGWLNKNPEKMVIAPEDWFRAVHTDIVPPSWIKM Amphritea WP_ 518450815 protein 26.76 MVIVRLIGGLGNQLFQYAYALSLLEQGYDVKLDASAFESYTLHGGFGLGEYAERLEVATTEEVDMVSRVGRIS 79 japonica 019621022.1 [Amphritea TLLRKLQGKKSRRVIKESNFSYDEKMLTPEDSHYLVGYFQSELYFNKIRGELLSALDLKHKLSPYTEASYLAIADA japonica] SVSVSMHIRRGDYVSDKAAHNTHGVCSLDYYYAAVTFFEERYPDVDFYIFSDDIEWVKENLNVQRAHYISSEE KRFAGEDIYLMSQCDHNIVANSSFSWWGAWLNANEDKIVVAPRQWYADSNMQRLSKTLVPDTWIRL Desulfovibrio YP_ 218887785 glycosyl 26.76 MRPVVVDIFGGLGNQMFQYAAAKSLAERLGVRLELDVSMFSGDPLRAFSLGEFAITDHVRGKSRSSLLVRFA 80 vulgaris; 002437106.1 transferase RSLGFGSSSKCVEPFFHYWEGINEIEAPVHMHGYWQSEKYFKAYEDLIRRTFSFSACEGVASSGKYAGVSSP Desulfovibrio family protein MSVSVHLRRGDYKEQKNVVVHGILGREYYDAAYSIIKQGCPSACFFVFTDAINEAVDFFSHWNDVLFVDGN vulgaris; [Desulfovibrio NQYQDMYLM SQCRHHIIANSSYSWWGAWLGAFSDGMTVAPKMWFAYDVLKEKSIKDLFPEDWIVL str. vulgaris str. ′Miyazaki F′ ′Miyazaki F′] Spirosoma WP_ 522095677 protein 26.76 MIISRITSGLGNQLFQYAVARHLSLKNKTSLYVDLSYYLYQYHDDTSRNFKLGNFSVPYHTLQQSPVEYVSKAT 81 spitsbergense 020606886.1 [Spirosoma KLLPNRSLRPFFLFQKERQFHFDEQILQSRAGCVILEGFWQSEAYFRDNADTIRRDLQLSGTPSPEFNQYRELI spitsbergense] RETPMSVSIHVRRSDYVNHPEFSQTFGFVGIDYYKRAIELARKELANPRFFVFSDDKEWSKTNLPLGEDSVFV QNTGLNGDVADLVLMSHCQHHIIANSSFSWWGAWLNPNAGKLVITPKNWYKNKPAWNTKDLLPPTWLS I Lachno- WP_ 511037988 protein 26.73 MNIIRMSGGIGNQMFQYALYLKLVSLGKEVKFDDVTEYELDNARPIMLSVFGIDYPKASREELVELTDASMD 82 spiraceae 016292012.1 [Lachno- FLSRVRRKIFGRKSGEYHEASADYDETVLEKEHAYLCGCFQSERYFKDIEYEVREAYRFRNVVVPEEIRGGIETY bacterium  spiraceae ERQIGESLSVSIHIRRGDYLDAADVYGGICTDAYYNQAIRYMIKKYENPSFFVFTNDTFWAEKWCEVRERETG 28-4 bacterium KRFTVIKGTDEETGYIDLMLMSRCKAHIIANSSFSWWGAWLDASPDKCVVAPVKWINTRECRDIYTEDMVR 28-4] IGSNGKISFSNCSSL Lachno- WP_ 511048325 protein 26.71 MVVVRIWEGLGNQLFQYAYARALSLRTKDRVYLDISEYEMSPKPVRKYELCHFKIKQPVINCGRIFPFVNKDS 83 spiraceae 016302211.1 [Lachno- FYTKNNQYLRYFPAGLIKEEDCYFKRDFCELKGLYLKGWFQSEKYFKEFESHIREEIYPRNKIKITRGLRKILNSD bacterium  spiraceae NTVSVHIRRGDFGKDHNILPIEYYENSKRVILERVDNPYFIIFDDILWVKENMNFGLNCFRYMDKEYSYKDYEE COE1 bacterium LMIMSRCKNIIANSTFSWWGAWLNPSKDKIVIAPKKWFLYNPKKDFDIVPNDWIRV COE1] Para- WP_ 492502331 alpha-1,2- 26.69 FutZB MKIVNIIGGLGNQMFQYAFAVALKAKYPNEEVFIDTQHYKNAFIKVYHGNNFYHNGYEIDKVFPNATLEPAR 84 bacteroides; 005867692.1 fucosyl- PKDLMKVSFYIPNQVLARAVRRIFPKRKTEFVTDQQPYVFIPEALSVIDDCFYDGYWMTPLYFDKYRDRILKEF Para- transferase TFRPFDTKENLELEPLLKQDNSVTVHIRRGDYVGSSSFGGICTLDYYRNAIREAYNLITSPEFFIFSNDQKWCM bacteroides [Para- ENMRNEFGDAKVHFIAHNRGADSYRDMQLLSIARCNILANSSFSWWGAYLNQRKNCFIICPHKWHNTLEY sp. 20_3; bacteroides] SDLYLPTWIKI Para- bacteroides distasonis CL09T03C24 Bacteroides WP_ 488624717 protein 26.62 MFVIRLIGGVGNQLFQYTFGQFLRHKFGVEVCYDIVAFDTVDKGRNLELQLLDESLPLFETSNFFFSKYKSWK 85 sp. HPS0048 002561428.1 [Bacteroides KRLFLYGFLLKKNNKYYTKYAPEEISLFTEKGLSYFDGWWQYPALLRDTINNMEDFFIPKQPIPVQIQKYYNEIL sp. HPS0048] LNNFAVALHVRRGDYFTSKYAKTYAVCNVEYYTSAVNLMCEKLRSCKFYVFSDDLDWVKSNLILPSNTVYVK NYDINSYWYIYLMSLCHHIIISNSSFSWWGATLNRNFHKIVIAPKYWSTKKNNTLCDNSWIKI Bacteroides WP_ 511013468 protein 26.58 MKIINILGGLGNQMFEYAMYLALKNAHSEEEILCSTRSFCGYGLHNGYELGRIFGIQVKEASLLQLTKLAYPFF 86 theta- 016267863.1 [Bacteroides NYKSWQVMRHWLPVRKTMTRGAINIPFDYSQVMREDSVYYDGYQWNEKNFLHIREEILTAYTFPKFDDEK iotaomicron; theta- NQELADIIVKSNAVSCHIRRGDYLKEINMCVCTSSYYAHAISYMNEEINPNLYCVFSDDIEWCRNNICELMGE Bacteroides iotaomicron] DKKIIFIDWNKGEKSFRDMQLMSLCKHNIIANSSFSWWGAWLNRNDKKIVVAPTRWIASEVKNDPLCDSW theta- KRIE iotaomicron dnLKV9 Desulfovibrio YP_ 78357918 glycosyl 26.56 MKFVGVWILGGLGNQMFQFAAAYALAKRMGGELRLDLSGFKKYPLRSYSLDLFTVDTPLWHGLPMSQRRF 87 alaskensis; 389367.1 transferase RIPMDAWTRGSRLPLVPSPPFVMAKEKNFAFSPIVYELQQSCYLYGYWQSYRYFQDVEDDIRTLFSLSRFATL Desulfovibrio [Desulfovibrio ELAPVVQLNEVESVAVHLRRGDYITDAASNAVHGVCGIDYYQRSMSLVRRSTTKPIFYIFSDEPEVAKKLFAT alaskensis alaskensis EDDVVVMPSRRQEEDLLLMSRCKHHIIANSSFSWWAAWLGKRASGLCIAPRYWFARPKLESTYFDLIPDE G20 G20] WLLL Prevotella ETD21592.1 564721540 protein 26.56 MDIVVIFNGLGNQMSQYAFYLAKRKSGSRCHCIFHNVSTGFHNGSELDKVFGIKYEKGIFSKLLSKIYDIFDGIP 88 oralis CC98A HMPREF1199_ KLRKKLNSLGIHIIREPRNYDYTASLLPRVSRWGLNYFVGGWHSEKYYTEILQEIKNTFSFKIDDEIKDIDFYEFYS 00667 LIHNDINSVSLHIRRGDYVGANEYSYFQFGGVATLEYYHKAIDEIYQRIENPTFYVFSDDIGWCKTTFLKNNFIF [Prevotella VDCNCGEKSWRDMFLISQCKHHIIANSTFSWWGAWLSIFHNSITICPKEFIKGVVIRDVYPDTWIKLSS oralis CC98A] Comamonadaceae YP_ 550990115 glycosyl- 26.54 MASKISKIIPRIFGGLGNQLFIYAAARRLALVNGAELALDDVSGFVRDHEYNRHYQLDHFNIPCRKATAAERLE 89 bacterium CR 008680725.1 transferase PFARVRRYLKRKWNQRLPFEQRKYLVQESVDFDERLLTFKPRGTVYLEGYWQSEDYFKDIEPQIRADLRIHPP [Comamon- TDTVNQQMAERIRATNAVAVHVRFFDAPAQSALGVGGNNAPGDYYQRAIKVMQEQAPDAQYYIFSDQP adaceae QAARARIPLRDDHVTLVNHNQCDAVAYADLWLISQCQHFIIANSTFSWWGAWLGKTPESIVIAPGFEKREG bacterium CR] AMFWGFRGLLPDRWVKL Vibrio WP_ 550250577 WblA protein 26.51 MKDSRIVKLNGGLGNQMFQFALAFALKKKLNVAVKFDTELLDTNRTEFKSLERFGLIVDKLTITEKFKYKGLE 90 nigripulch- 022596860.1 [Vibrio SCKYRKICNWISNFTTINIHKGYYKEKERGVYDRGIFDSNVKYIDGYWQNQEYFNDFRSELLNKFNLNGKVSN ritudo; nigripulch- HAIQYLKEITSVQNSVSIHVRRGDYLLLDVYRNLTLDYYSEAIKLVRITNPDSKFFIFSNDINWCKSNFKSVDNAI Vibrio ritudo] FVDSTVDEFDDMFLMSKCKTNIIANSTFSWWAAWLNNSGKIVYCPKKWRNDTTENHKGLPEGWNIIDK nigripulch- ritudo AM115; Vibrio nigripul- chritudo Pon4; Vibrio nigripulch- ritudo SO65 Sulfuro- YP_ 268680406 glycosyl- 26.48 MIIIKIMGGLTSQMHKYALGRVLSLKYNVPLKLDLTWFDNPKSDTPWEYQLDFNINATIATVSEIKKLKGNN 91 spirillum 003304837.1 transferase LFNRIARKIEKFFSIRIYKKSYINKSFISISDFHKLKSDIYLDGEWNGFKYFEDYQDTIKNELTLKRGSSINIQNTIE deleyianum; family protein LKSSDNSVFLHIRRGDYLSNKNAAAFHAKCSLDYYYKAIQIVKEKIDNPIFYISDDILWVKKNFVINESCRFME Sulfuro- [Sulfuro- KNQNFEDLLLMSYCKHGITANSGFSLMAGWLNQNKDKMIIVPQTWVNDDRININILNSLEQDNFTIIR spirillum spirillum deleyianum deleyianum DSM 6946 DSM 6946] Escherichia WP_ 486318742 glycosyl 26.47 MTFIVRLTGGLGNQMFQYALARSLAKKYNARLKLDISYYHNQPHKDTPRTFELNQLCIVDNILNSSSFSEKFLY 92 coli; 001581194.1 transferase IYDKLRVKLSKKISLPYFRNIVTPVNFNCIDFAEDKDYYFLGHFQELSNIYSIDESLRSEFKPNQEIMNLAHQSKIY Escherichia 11 family ELIKQSRGSVALHIRRGDYVTNKNAAEHHGVIGLSYYVNALSYLENVSEFFDVFVFSDDPEWARKNIKNSRNL coli Jurua protein FFCDEGNCRYSKKYSTIDMYLMSQCDHFIIANSTYSWWAAWLGNYPSKHVVAPARWNANNSPYPILQNW 18/11: [Eschericha KAIHE Escherichia coli] coli 180600; Escherichia coli P0304777.1; Eschericha coli P034777.2; Eschericha coli P034777.3; Eschericha coli P0304777.4; Eschericha coli P0304777.7; Eschericha coli P0304777.9; Eschericha coli P0304777.10; Eschericha coli P0304777.11; Eschericha coli P0304777.12; Eschericha coli P0304777.13; Eschericha coli P0304777.14; Eschericha coli P0304777.15 Firmicutes wP_ 547109632 protein 26.44 MVGVQLSGGLGNQMFEYALYLKLKSMGKDVRIDDVTCYGAQEKQRVNQLSVFGVSYEHMTKQEYEQITD 93 bacterium 021914998.1 [Firmicutes SSMSPLHRARRLLCGRKDLSYREASCNYDPEILRREPALLLGYFQTERYFADIKDQVREAFTFRNLTLTKESAA CAG:24 bacterium MEQQMKECESVSVHIRRGDYLTPANQALFGGICDLDYYHRAVAEIRKRKPDVKFFLFSNDMEWTKEHFCGS CAG:24] EFVPVEGNSEQAGEQDLYLMSCCKNHILANSSFSWWGAWLDNGKDKLVIAPEKWMNGRGCCDIYTDEMI RV Amphritea WP_ 518452719 protein 26.42 MVKIKIIGGLGNQMFQYAAAKSLAVLNNTRVSANVSVFSNYKTHPLRLNKLNCDCEFDFTRDFRLVLSGFPLL 94 japonica 019622926.1 [Amphritea GSAFSKKSMLLNHYVEKDLLFDSSFFLDLDNVLLSGYFQSEKYFSNIRELLIQEFSLDDRLTEAELAINNKIESCN japonica] SIAIHIRRGDYITDLSANNIHGICSEEYFEKALNYLDSINVLSDPTTTLFIFSDDILWCKDNLAFKYRTVFVEGSVD RPEVDIHLMSKCKHQVISNSTFSWWGAWLNTNLDKCVIAPLKWFNSLHDSTDIVPKQWMRL Bacteroides WP_ 492689153 protein 26.41 MKQTIIMSGGLGNQMFQYALYCSMREKGIRVKIDISLYEFNRMHNGYMLDYAFGLNISHNKINKYSVLWTR 95 salyersiae; 005923045.1 [Bacteroides LIRSNRAPFLLFREDESRFCDDVFTTYKPYIDGCWIDERYFFNIKKKIISQFSFHNIDQKNLMVANMMKVCNS Bacteroids salyersiae] VSLHIRRGDYLSQSMYNICNESYYKSAIEYIISRVEDSKFFIFSDDPEWCKYFMEKFNVDYEIIQHNFGKDSYKD salyersiae MYLMTQCKHNIIANSTFSWWGAWLNNNAGKNVVCPSVWINGRDFNPCLEEWYHI WAL10018 =  DSM 18765 = JCM 12988 Bacteroides WP_ 492241663 protein 26.38 MDIILLHNGLGNQMSQYAFYLSKKKNGIHTSYICLSNDHNGIELDKVFGVECQMGCKKIFLLFILRLLMSNRT 96 fragilis; 005786334.1 [Bacteroides GFLIRKVNLLFSKIKIKLITENLDYSFHPSFLSASPYCLAFWVGGWHHPQYYSEISSQIKEAFTFKRSLLDERNICI Bacteroides fragilis] EKRMREPNSVCLHIRRDGYLTGINYELFGKVCNEQYYQKAIDYIEGKLSDICYYVFSNDMEWAKKILLGKNAV fragilis FVDWNRGEESWKDMYLMSKCNLIIPSNSTFSWWAWLCEHPVNIVCPKLFVYGDEQSDIYLDNWHKIE CL03T00C08; Bacteroides fragilis CL03T12C07 Bacteroides WP_ 494751435 protein 26.37 MMGIEKTNMVIVRLWGGIGNQLFQYSFGEFLREDYQVDVIYDIASFGKSDKLRKLELSVVVPGIPVTTDISFSK 97 nordii; 007486843.1 [Bacteroides YVGTKNRLLRFIYGLKNSFIEEKYFSDEQLFKYLSKRGDVYLQGYWQKTIYAETLRRKGSFFLSQEEPIVLHTIKA Bacteroides nordii] KIQEAEGAIALHVRRGDYFSSKHINTFGVCDAHYYEKAVDIMRGRVSNAMIFVFSDDLDWVRRYVNLPTNVI nordii  YVPNYDIPQYWYIYLMSLCRHNIISNSSFSWWGAFLNMNTNKIVVSPSKWTLNSDKTIALDEWFKI CL02T12C05 Butyrivibrio YP_ 302669783 glycosyl 26.37 MECSMIIIKFCGALGNQLFQYALYEKMRILGKDVKADISAFGDGNEKRFFYLDELGIEFNIASADEIAEYLNRKT 98 proteo- 003829743.1 transferase IRFVPGFLQHRHYYFEKKPYVYNKKILSYDDCYLEGYWQNYRYFDDIKDELLKHMKFPCLPLEQKKLAEKMEN clasticus; 11 ENSVAVHVRMGDYLNLQDLYGGICDADYYDRAFSYIEGNISNPVYYGFSDDVDKASALLAKHKINWIDYNSE Butyrivibrio [Butyrivibrio KGAIYDLILMSKCKNNIIANSSFSWWGAYLEYNNGKVVVSPNRWMNCFENSNIAYWGWISL proteo- proteo- clasticus clasticus B316 B316] Prevotella YP_ 294674032 family 11 26.33 MRIVKVLGGLGNQMFQFALYKALQKQYFEERVLLDLHCFNGYHKHRGFEIDSVFGVTYEKATLKEVASLAYP 99 ruminicola; 003574648.1 gylcosyl YPNYQCWRIGSRILPVRKTMLKEEPNFTLEPSALSLPDSTYYDGYWQHEEYFMHIREEILSTYAFPAFDDERN Prevotella transferase KTTAQLAASTNSCSIHIRRGDYLTDPLRKGTTNGNYVIAAIKEMQQEVKPEKWLVFSDDIAWCQQHLASTLD ruminicola [Prevotella ATNTIYIDWNTGANSIHDMHLMALCRHHIIANSSFWWGAWLSQQDGITIAPSNWMNLKDVCSPVPDN 23 rumincola 23] WIKI Prevotella WP_ 494223898 protein 26.33 MKIIKIIGGLGNQMFQYALAIALQQQYKDEEIRLDLNCFRGYNKHQGYLLDEIFGRRFRAASLQEVARLAWPY 100 salivae; 007135533.1 [Prevotella PHYQLWRVGSRVLPRRQTMVCEPADGSFSPDVLTLEGNRYYDGYWQDERYFKAYRKEIIEAFKFSPFVGDG Prevotella salivae] NRHVENMLRNERFASLHVRRGDYLNDALYQNTCGIDYYQRAISQMNAMNANPSCYFIFSDDIAWCKTHIEPL salivae CEGHRPYYIDWNKGKEAYRDMQLMALCKYHIIANSSFSWWGAWLNDAEDGITIAPQQWYSHGNKPSPAS DMS 15606 ESWIKV Lachno- WP_ 511045640 protein 26.3 MNIVRISDGLGNQMFQYAYARKISILSRQRTYLDIRFINNEDLVKKGNHVQFRKKLGHRKYGLSHFNVSLQIA 101 spiraceae 016299568.1 [Lachno- DLKMLSHWEYLIQSNCMQQLIYSLSMQDKWIWRYRHEEVNYDGMLSKVELLFPTYYQGYFFALKYYDDIKH bacterium spiraceae ILQHDFSLKDKMKLLPEDRDALYNRNTISLHVRRGDFLEINRDISGSEYYEKAVQMIGSKVESPIFLIFSDDIEW COE1 bacterium VHEHIRIPNDKIYVSGIGYEDYEELTIMKHCKHNIIANSTFSYWAAYLNSNKDKIVICPKHWRERIIPKDWICI COE1] Bacteroides WP_ 495118115 alpha-1,2- 26.28 MIVVNVNAGLANQMFHYAFGRGLEAKGWNIFYDQTNFKPRKEWSFENVQLQDAFPNLGLKMMPEGKFK 102 dorei; 007842931.1 fucosyl- WICNVVTNKLSKGLHLAMINLHNLIGDEKYEFETTYGYDPDIEKEITKNCILKGFWQSEKYFAHCKDDIRKQFS Bacteroides transferase FLPFDEEKNIVIMNKMVKENSVAIHLRKGADYLDSELMGKGLCGVEYYIKAIEYIKKNIDNPVFYVFTDNPVW dorei 5_1_ [Bacteroides VKNNLPKFDYILVDWNEVAGKKNFRDMQLMSCAKHNIIANSTYSWWGAWLNPNPNKIVIGPAKFFNPIN 36/D4 dorei] NFFSSSDIMCEDWVKI Roseobacter WP_ 495485361 alpha-1,2- 26.28 MLSKDPGMITTRLHGRLGNQMFQYAAGRALAARLGVPLADSRGAKLRGEGVLTRVFDLPLAQPLSLPPLK 103 sp. SK209-2-6 008210047.1 fucosyl- QDAPLRYAAWRLTGRPFRFRREQGLGYNPAFETWGDDSYLHGYWQSEAYFDSIADQIRQDFTFPEFSNSQ transferase NREMAQRIAGSTAISLHVRRDGYVALAAHVLCDQAYYEAALTRILEGVEGSPTVYVFSDDPNWAKENLPLPC [Roseobacter EKVVVDFNGPDTDFEDMRLMSLCQHNIIGNSSFSWWAAWLNTHNEKRVAGPAHWFGNPKLQNPDILPE sp. SK209-2-6] SWLKISV alpha WP_ 518900826 protein 26.26 MIYSRIRGGLGNQLFQYCVARSLADNLGTSLGLDVRDFNENSPYLMGLKHFNIRADFNPPGMIEHKKNGYF 104 proteo- 020056701.1 [alpha RYLIDVVNGKQKFVYKEPHLNFDKNIFSLPSNNYLKGYWQTEKYFIKNKVNILNDLKIISHQSDKNKTISSKIAN bacterium proteo- NTSVSLHIRRGDYISNSAYNSTHGTCSLAYYTNAVNFLVNKIGGNFKVFAFSDDPEWVSSNLKLPVDICFVKN SCGC AAA076- bacterium NSSEYJYEDLRLMSECNHNIIANSSFSWWGAWLNTNHNKTVITPCKWYADNSTKNADITPSNWIKI C03 SCGC AAA076- C03] Helicobacter wP_ 490188900 protein 26.26 MGGGGQDLRLFELMLYNISLPLCFDYKTLVKYFYSNDKSLKYNFPLQYIRATRSKYHKLYWLALKHYKYFYDE 105 bilis; 004087499.1 [Helicobacter DPQGDNIVKMYLNNSLEKHAYPFGYFQNLIYFDEIDSIIREEFCLKIPLKPHNQALKEKIEKTENSVFLHVRLGD Helicobacter bilis] YLKMEATDGGYVRLGKEYYQSALEILKTRLGQPHIFIFSNDIEWCEKNLCNLLDFTGCHIEFVKANGEGNAAE bilis WiWa EMELMRACKHAVIANSTFSWWASYLIDNPDKQIIMPTQVFNDTRRIPKSNMLAKKGYILIDPFWGMHSIV Ralstonia sp. WP_ 498513378 glycosyl 26.26 MIVTRVIGGLGNQMFQYAAGRALARRLGVPLKIDSSGFADYPLHNYGLHHFALKAVQAGDREIPSGRAENR 106 GA3-3 010813809.1 transferase WAKALRRFGLGTELRVFRERGFAVDPEVMKLPDGTYLDGYWQSESYFAEMTQELRRDFQIATPPTSENAE family protein WLARIGGDEGAVSIHVRRGDYVTNASANANHGICSLDYYMRAARYVAENIGVKPTFYVFSDDPDWVAGNL [Ralstonia HLGHETRYVRHNDSARNYEDLRLMSACRHHIIANSTFSWWGAWLNASEKKVVIAPAQWFRDEKYDTRDLL sp. GA3-3] PPTWTKL Bacteroides WP_ 490431888 protein 26.25 MVVVYIAAGLANKMFQYAFSRGLMSHGLDVFLDQTSFQPEWSFEDIALEEVFPNIEIKNAPNNMFSLAYKK 107 ovatus; 004303999.1 [Bacteroides DLLSRIYRRMSAFFPNNRYLMERPFIYDELIYKKATNNCIFCGLWQTELYFNFCERDVRRNFVFTPFQDDQNI Bacteroides ovatus] KLAEKMKNENSVAIHIRKGADYLKRNIWDGTCSVEYYNQAINYLKEHVSNPVFYLFTDNPEWVEENLKNIDY ovatus 3_8_ KLVDWNPVSGKQSYRDMQLMSCAKHNIIANSTYSWWGAWLNNNPQKIVVAPKIWFNPKIEKAPYIIPDR 47FAA WIRL Loktanella WP_ 518799952 protein 26.23 MIITKLIGGLGNQMFQYAAGRSLAMRHGVPLLLDITELRSYPKHQGYQFEDVFAGRFEIAGLIPLIRVLGRKAR 108 vestfoldensis 019955906.1 [Loktanella KVPKTVAVVSPKWPPMGDHVWVRQRTHDYDAAFESIGADCYLSGFWQSEKYFATIAPQIRESFRFKEALTG vestfoldensis] ANAAIASRMKEAPSAAIHIRRGDYVTDKGAHAFHGLCAWDYYDAAIDHISRHEPDARFFVFSDDVVAAQER FANRQRAEVVAVNSGRHSYRDMMLMAQCKHQIIANSTFSWWAAWLNQNPDKIVVAPGTWFSGNDGQI KDIYCKDWIVI Flavobacter- WP_ 515558304 protein 26.14 MDVVIIFNGLGNQMSQYAFYSQKKKINNSTYFVPFCKDHNGLELETVFSLNTKETLIQKSYILFRILLTDRLKIV 109 ium sp. 016991189.2 [Flavobacter SDPLKWILNLFKCKIVKESFNYNYNPEYLKPSKGITFYYGGWAEKYFAKENQQIKSVFEFTGDLGKINKEHVK ACAM 123 ium sp. DIASTNAVSLHVRRGDFMNEANIGLFGGVSTKAYFEGAIKLIATKVDHPHFFVFSNDMDWVKENLSMDTVT ACAM 123] YVTCNSGKDSWKDMCLMSLCQHNIIPNSTFSWWGAWLNKNPHKIVVCPSRFLNNDTYTDIYPDSWVKISD Y Bacteroides WP_ 492219620 glycosyl 26.1 MMKLVRMTGGLGNQMFIYAFYIQMKTIFPELRIDMSEMKKYKLHNGYELEDVFSIRPQTISAHKWLKRVIV 110 fragilis; 005779407.1 transferase YAFFSIIREKSEEELSIHKYTQHKRWPLVYYKGFFQSELFFKESSDTIRDIFSFNTENANFRTKEWAKIIKEQRSSV Bacteroides family 11 SIHIRRGDYTSAKNKIKYGNICTEEYYQKAISIILKKEPKAFFHIFSDDVEWTKAHLKIHHLPHQYISWNKGPDS fragilis [Bacteroides WQDMMLMSLCRHNIIANSSFSWWGAWLNAYKDKTVIAPSRWSNVKKTPHILPESWISISI 3_1_12 fragilis] Spirosoma WP_ 522086793 protein 26.09 MIISRVTSGLGNQLFQYAAARSLSLRNKTAFYVDLSYYLYEYPDDTSRSFKLGFFSVPYRILQESPVEYLSKSTKL 111 panaciterrae 020598002.1 [Spirosoma FPNRSLRPFFLFLKEKQFHFDPTILQAHAGCVIMEGFWQSECYFRDHAEIIRRELQLSKSPSSEFEGYHQQIQA panaciterrae] TPVPVSVHVRRGDYVNHPEFSKTFGFIGLDYYKTAIRHLTKTIKNPHFYVFSDDKEWARANLPLPTDSVFVTN TGPSGDVADLVLMSTCHHHIIANSSFSWWGAWLNPNPDKLVITPKLWYKNQPTWNTKDLLPPTWSVSL uncultured EKE06672.1 406985982 glycosyl 26.09 MIITKLTGGLGNQLFQYAIGRNLIYINGSDLKLDVSEYDVSNKGNFRHYALDKFNTIQNFASKKETNNFKFGVF 112 bacterium transferase KKWLYKSGIVKNKNYFLEKKFNFDKEILKIKDNAFLQGYWQSEKYFIGIRDILLQEFSLKENIELKFGEILKEINES family 11 NSVSIHVRRGDYVKNPKNLSFHGVCSPKYYSESTSKIASLIEKPVFFVFSDDIEWVKENLNITFPVVYLSGIKNIK [uncultured SYEELVLMSKCKHNIIANSSFSWWGAWLNTNQKKIVIAPKRWFNDVKLDTTDLIPENWIRI bacterium] Thermo- NP_ 22298537 alpha-1,2- 26.07 MIIVHLCGGLGNQMFQYAAGLAAAHRIGSEVKFDTHWFDATCLHQGLELRRVFGLELPEPSSKDLRKVLGA 113 synechococcus 681784.1 fucosyl- CVHPAVRRLLAGHFLHGLRPKSLVIQPHFHYWTGFEHLPDNVYLEGYWQSERYFSNIADIIRQQFRFVEPLDP elongatus; transferase HNAALMDEMQSGVSVSLHIRRGDYFNNPQMRRVHGVDLSEYYPAAVATMIEKTNAERFYVFSDDPQWVL Thermo- [Thermo- EHLKLPVSYTVVDHNRGAASYRDMQLMSACRHHIIANSTFSWWGAWLNPRPDKVVIAPRHWFNVDVFD synechococcus synechococcus TRDLYCPGWIVL elongatus elongatus BP-1 BP-1] Colwellia WP_ 517858213 protein 26.03 MKIVKIAGGFGNQLFQYAFYLALDKKYAEQVCLDSLDMAKYRLHNGYELEGIFKLDARYCTEEQRIIVRKDNN 114 piezophila 019028421.1 [Colwellia IFTKLLSSLKKKLGNNKNYILEPKQEHFTFHEKSFGQANTPTYYKGYWQDVKYLENIEEELKSSLVFPEFELGKNI piezophila] ELANFISSNSSVSLHVRRGDYVQHKAFGGICDLSYYQRAVEQINTLVKDPIFIVFSDDIQSCKDNLNLEKAKFV DWNIGENSFRDMQLMTLCKHNIIANSSFSWWGAWLNANDDKNVICPDKWVHYTSATGVLPSEWIKIKAS V Prevotella WP_ 518810840 protein MKIVKIIGGLGNQMFQYALAIALQERWKDEEIKLDLHGFNGYHKHQGYQLDMLFGHRFEAATLTDVAQLA 115 maculosa 019966794.1 [Prevotella WPYPHYQLWRVGSRLLPKRRSMLCEPSKGLLPSDVLKQKGSLYYDGYWQDERYFRAIRPQIMAAFKFPDFT maculosa] DRRNLETEKRLKASEAVSIHVRRGDYLDDVLFQGTCNIAYYQRAIARLCQLKTPVFCIFSNDMAWCKVHIEPL LHGKEILYVDWNRGKESYRDLQLMTLCRHHIIANSSFSWWGAWLSKAEDGITIAPRHWYAHDAKPSPAAE RWIKV Salmonella YP_ 525860034 fucosyl- 25.99 MYSCLSGGLGNQMFQYAAAYILKQYFQSTTLVLDDSYYYSQPKRDTVRSLELNQFNISYDRFSFADEKEKIKLL 116 enterica; 008261369.1 transferase RKFKRNPFPKQISEILSIALFGKYALSDRAFYTFETIKNIDKACLFSFYQDADLLNKHKQLILPLFELRDDLLDICKN Salmonella [Salmonella LELYSLIQRSNNTTALHIRRGDYVTNQHAAKYHGVLDISYYNHAMEYVEREGKQNFIIFSDDVRWAWKAFL enterica enterica ENDNCYVINNSDYDFSAIDMYLMSLCKNNIIANSTYSWWGAWLNKYEDKLVISPKQWFLGNNETSLRNAS subsp. subsp. WITL enterica enterica serovar serovar Worthington Cubana str. str. ATCC  CFSAN002050] 9607;  Salmonella enterica subsp. enterica serovar Cubana str. CFSAN001083; Salmonella enterica subsp. enterica serovar Cubana str. CFSAN002050; Salmonella enterica subsp. enterica serovar Cubana str. CVM42234 Bacteroides WP_ 495935021 protein 25.94 MKKVIFSGGLGNQMFQYAFYLFLKKKGIKAVIDNSLYSEFKMHNGFELIKVFDIKESIYRTYFLKVHLIFIKLLMK 117 sp. 3_2_5 008659600.1 [Bacteroides IPPVRKLSCKDDVIPIGDHEFDPPYARFYLGYWQSKKIVNYVIEELRAQFIFRNIPQMTIEKGDFLSSINSVSIHIR sp. 3_2_5] RGDYMGIPAYQGICNEIYYERAISFMKEHFLNPRFYVFSNDSIWAKLFLEKFDIDMEIIVTPPIYSYWDMYLMS RCRNHIIANSTFSWWAAVLNINKDKIVISPTIFKKDECIDIIFDDWVKISNI Clostridium WP_ 547662453 protein 25.86 MIMLQMTGGMGNQMFTYALYRSLRQKGKEVCIEDFTHYDTPEKNCLQTVFHLDYRKADREVYQRLTDSEP 118 CAG:510 022124550.1 [Clostridium DFLHKVKRKLTGRKEKIYQEKDAIIFEPEVFQTDDVYMIGYFQSGRYFEKAVFDLRKDFTFAWNTFPEKAKKLR sp. CAG:510] EQMQAESSVSLHIRRGDYMNGKFASIYGNICTDAYYEAARRYMKEHFGDCRFYLFTDDAEWGRQQESEDT VYVDASEGAGAYVDMALMSCCRHHIIANSSFSWWGAWLDENPDKTVIAPAKWLNISEGKDIYAGLCNCLI DANGSVQGE Rhodopirellula WP_ 495940880 glycosyl 25.86 MIVTRLIGGLGNQLFQYAFGHSLARSTYQTLLIDDSAFIDYRLHPLAIDHFTISASRLSDADRSRVPGKFLRTPV 119 europaea; 008665459.1 transferase GRALDKVSRFVPGYQGVLPVRREKPFGFRESLLARESDLYLDGYWQSEKFFPGLRGSLREEFQLREQPSETTR Rhodopirellula family protein RLSAQMKSENSVAIHVRRGDYVTSAKAKQIYRTLDADYYRRCLLDLAAHETDLKLYLFSNDVPWCESNLDVGI europaea [Rhodo- PFTPVQHTDGATAHEDLHLIAQCRHVVIANSTFSWWGAYLGQLHPTRRVYYPEPWFHPGTLDGSAMGCD SH398 pirellula DWISEASLEEQSSLKSSRRAA europaea] uncultured EKD23702.1 406873590 glycosyl 25.82 MIIVKLKGGMGNQMFQYAIGRNLATKLGTQLRLDLTFLLDRSPRKDFVFRDYDLDIFALDVAFAGPTDLKPFT 120 bacterium transferase QFRISHLTKIYNIFPRLLGRPYVISEPHFHFSEAILKSSDNVYLDGYWQSEKYFKEIENSIRDDFKFRQPLEGRAA family protein EMAAQIKNEDRAVCLNVRRADFVTSKKAQEFHGFIGLDYYQKAVDLLVSKVGPLHLFIFSDDVDWCAANLK [uncultured FNYPTTFVTKDYSGKKYEAYLQLMTLCRHYIIPNSTFAWWGAWLNSDPNKIVIAPKQWFKEASISTTDIIPST bacterium] WIRL Bacillus WP_ 446510160 protein 25.74 MIIVKLKGGLGNQMFQYALGKSLAYYDKPLKIDADYIKNNEGYVPRDFSLSKFNIELDLYQEADKERVGFILK 121 cereus; 000587678.1 [Bacillus NNFLAKKLRNYFLKKGKYKGKYIIENPDNLGLFKKELFENHNESMYIDGYWQSYLFNNIRECLIKEFNLKPEYT Bacillus cereus] KEMTEIMQRINETNSVAVHIRRGDYVKLGWTLDTTYYKKAIAEIVKNVDNPKFYVFSDDTDWVRSNLQELD cereus AH1271 NAVFIGECNLFDYQELWLMSTCKHHNIISNSTFSWWGAWLNQNDHQVVVSPSAWINGMSVETTSLIPDSW KRV Firmicutes WP_ 548309386 protein 25.74 MDIIRMEGGLGNQLFQYALYRQLQFMGRTVKMDVTTEYGREHDRQQMLWAFDVHYEEATQEEINRLTD 122 bacterium 022499937.1 [Firmicutes GFMDLPSRIRRKLTGRRTKKYAEADSNFDPQVLLKTPVYLTGYFQSEKYFKDVEGILHTELGFSDRIYDGISEVF CAG:95 bacterium ADQIRNYQKQIRETESVSLHVRRGDYLEHPEIYGMSCTMEYYQAGVRYIRERHPDAEIFVFTNDPVFTEKWL CAG:95] QENFLGDFTLIQGTSEETGYLDLMLMSQCKHQIMANSSFSWWGAWLNPNKDKIVVAPEPWFGDRNFHDI YTEEMIRSPRGEVKKHG Prevotella WP_ 490508875 alpha-1,2- 25.74 MIAATLFGGLGNQMFIYATVKALSLHYQVPMAFNLNHGFANDYKYHRKLELCKFNCQLPTAKWITFDYRGE 123 oris; 004374901.1 fucosyl- LNIKRISRRIGRNLLCPNYQFVIEEEPFHYEKRLFEFTNKNIFLEGYWQSPCYFENYSKEIRADFQLKVPLSKEML Prevotella transferase EEIYALKATGKTLVMLGIRRYQEVEGRDICTYKLCDEKEYYIKAITYIQERIPNALFVVFTQDKEWATTHLPKGAE oris F0302 [Prevotella FYFVKDKQDEYATVADMFLMTQCTHAIISNSTFYWWGAWLQCTTKNHIVIAPDSFINSDCVCKEWIILKRNS oris] LC Escherichia AAO37719.1 37528734 fucosyl- 25.73 MYSCLSGGLGNQMFQYAAAYILQRKLKQRSLVLDDYFLDCSNRDTRRRFELNQFNICYDRLTTSKEKKEISII 124 coli transferase RHVNRYRLPLFVTNSIFGVLLKKNYLPEAKFYEFLNNCKLQVKNGYCLFSYFQDATLIDSHRDMILPLFQINEDL [Eschericha LHLCNDLHIYKKVICENANTTSLHIRRGDYITNPHASKFHGVLPMDYYEKAIRYIEDVQGEQVIIVFSDDVKWA coli] ENTFANQPNYYVVNNSECEYSAIDMFLMSKCKNNIIANSTYSWWGAWLNTFEDKIVVSPRKWFAGNNKSK LTMDSWINL Leeia WP_ 516890767 protein 25.71 MIIVKIIGGLGNQMMQYAFAHACAKRLGVPFKLDTAFESYKLWPYGIHNFHTAPIASIEEIEHAKSMGVITE 125 oryzae 018150480.1 [Leeia TSFRRDDSLVSAVKDGMYIQGYWADYRYSESVWGELKPVFTLMDPLTPEQQALAMNISAPNAVAIMVKR oryzae] GDYVRNPNCFLLPQQYYRDAIKLVLDQQPDAVIYCFSQDDWVIAMLIIPAPKWVRGQGIDNGFVDMIL MSKARHRIVANSTRSIWASRWDQDGLTIVPSQFRRKDDPWLLQVYGPVLQPCYPPQWRWDVTGDGKKE AEMTSTALLQIAGGDVRGRKLRIGVWGFYEEFYQNNYIFLNKNAPIGHELLKPFNQLYQYGQAHNLEFVTLDL VADLSTLDAVLFFDAPNMRSPLVSSVMQLDIKKYLCLLECELIKPDNWQQSLHELFTRIFTWHDGLVDNMYI KYXQLMPWIESAOSLTAPFTETAKKGYLQKKLICWISGNKLVSHPFELYSKRIEVIRWRESHHPEHFDLYG MGWSASQYPSYKGKIDDKIVLXGYRFSLCYFNAKFLPGYITEKIIDCFKAGWPVYBCGAPNIAQWIPUNCFID SGKDTDALYTYLISMTEEVHADYLENIRQRRLGGKAYPFSADAFINMTRTIVQDCLFPHERTDVSWVPNY NHGNFWSAITSALNQNVSVELLVLDNASTDDSWSQLQFFADYPQVRLIRNRWNIGVQHNWNHATWLAT GRYVVMLSADDLLLPGHLEQAVKHLDHNPASSIYYTPCLWINEHDQPLGTLNHPGHLESDYVGGRDCISDLL KRDSYTPSAAVIRRETLNRIGSMNLHLKGAIDWDLWIRIAEISPAFIFRKQPGVCYRQHSGNNSVDFYASTAP LEDHIRIVESIIDRKVAVKYLLXAKEEIIAHLDNRMSYPENQIQHLLSRINNIKDYLRKGAGPVISVIIPTICNRPGI IAIMAIFSITYTTFKDFTVVHNDGGCDIGGIVDRFSDQLQISYVRSSQSGGAAASRNRALKLAKGRIIAYLDDD DVYLDSHLEKLVDAYKKKSFKHYINTYLIQERKEGRUELGRERRYAGISYSRAALLVSNRIPTPTWSHTKCLI DTIGDFDESLEILEDWDFLLRASKVTEFYQVNAIIVIVKSDKSRDUHTIRANADKLLAYHQKIYAKHPVINISI LANRQSLINSLSNRQDNPKNENSYQGWVNARQPNELAVQIIJIFRMMLOWSQYQFMIVMVVKQSQQ NLIANTIDSFCQQLYSGWLIVISDFSAPDESFINNEVLGWLTLETVEDENLLTQAFNGVLAEVPSQWVILPV GTRLTSTALLKVGDRIILNGGACVIYTDHDYVSDUGMIKDPVLKPAFNLDMLRSQDYIGSSIFFRTDSIAAVG GFASFPGARTYEACFRMLGNYGPQTIEHLPEPVMTrPENQPENSLRVAAMQLALEEHLHRNNISASIEEGYV TGTFLVQYHHSEQPRVSMIPNKDKHEFLAPCIETLMKVTQYPARCVIIVDNQSTDPDTLIYYEMESRFANNVK VIQYDNPFNFSAQCNLGAESATQDRILRLNNDTEIVCIILERMMQMAQRNDVGVVGARLVFPETVTIQH AGIVLGGKYPDEVFQFPYMNFPVDKDVSLNRTKVVQNYSAVRGACLLVRKSLYQQVGGMNEQNLAVLYGD VDLCLRIRQLHKSWWTPPSTLVHHTGKRLNSNSQHHKHLMMVIQTRQEREYMLSHWLDIIANDPYYHRLL DKSECNGTIDCTHTPLWDDIPSARPRLQGMALVGGSGPYRVNIVIPFHILERSAIAIMSNFCRRSKARLPSITEL ARNAPQVFWQNALADEFIRMLCMYKKYLPSVFRIQMLDDLLTElPDASSFKRHRQKNWRDAKARLRKSLKF CDRLIVSTFPLRTFAEDMIDDIIVVPNMLERSVWGDLVSKRRAGKKPRVGWVGAQQHAGDLALMTDWKA TGHEVDWVFCKGMCPDIRPYVAFVNTRWLTYDKYPQGIAALNLDLAIAPLEINAFNEAKSNLRLLEYGALG WPVICTDIYPYQTNNAPVCRVPNDASAWIEARSHIADUGATAUXILRQWVHDHYMIFDHAQFWLSA LTRPAGK Desulfovibrio WP_ 492830219 Glycosyl 25.68 MFQYAAARALSLRHSASLAADLTWFSQQFDVQTTPREYALPAFRLNLPEADKRIVATFRLNPTELRIVSFLRH 126 africanus; 005984173.1 transferase RICFPSRFLPRHITELSFDYWDGFRDILPPAYLDGYWQSERYFSDYPDIIRADFSMLSISEQAAWMSAKIASVQ Desulfovibrio family 11 DSISLHIRRGDYVNSLATRKAHGIDTERYYAKALEWIADRIGAATIFAFSDDPRWVRANFDFGKHKGIVVDGS africanus [Desulfovibrio WTAHEDMHLMSLCSHHIIANSSFSWWGAWLSTSQGITIAPKSWFSNPHIWTPDVCPATWERIPC PCS africanus] Akkermansia WP_ 547786341 Glycosyl 25.66 MAKGKIIVMRLFGGLGNQLFQYAFLFALSRQGGKARLETSSYEHDDKRVCELHHFRVSLPIEGGPPPWAFRK 127 muciniphila 022196965.1 transferase SRIPACLRSLFAAPKYPHFREEKRHGFDPGLAAPPRRHTYFKGYFQTEQYFLHCREQLCREFRLKTPLTPENARI CAG:154 family 11 LEDIRSCCSISLHIRRTDYLSNPYLSPPPLEYYLRSMAEMEGRLRAADAPQESLRYFIFSDDIEWARQNLRPALP [Akkermansia HVHVDINDGGTGYFDLELMRNCRHHIIANSTFSWWAAWLNEHAEKIVIAPRIWFNREEGDRYHTDDALIP muciniphila GSWLRI CAG:154] Dysgonomonas WP_ 493897667 protein 25.66 MKIVKLQGGLGNQMFQYAIARTLETNKKKDIFLDLSFLRMNNVSTDCFTARDFELSIFPHLRAKKLNSLQEKF 128 mosii; 006843524.1 [Dysgonomonas LLSDRVRYKFIRKIANINFHKINQLENEIVGIPFGIKNVYLDGFFQSESYFKHIRFDLIKDFEFPELDTRNEALKKTI Dysgonomonas mosii] VNNNSVSIHIRRGDYVHLKNANTYHGVLSLEYYLNCIKRIGEETKEQLSFFIFSDDPEYASKSLSFLPNMQIVD mosii DSM WNLGKNSWKDMALMLACKHHIIANSSFSWWGAWLSERGITYAPVKWFNNESQYNINNIIPSDWVII 22836 Prevotella WP_ 490506359 glycosyl 25.66 MDIVLIFNGLGNQMSQYAFYMSKKKFVPQSKCMYYKGASNNHNGSELDKLFDIKYSETFFCKLILLLFKLYENI 129 oris; 004372410.1 transferase PRLRKYFHILGINIVSEPQNYDYNESILKKKTRFGITLYKGGWHSEKYFLANKQDVLNTFSFKIAKEDKNFIDLAK Prevotella family 11 SIEEDTNSVSLHVRRGDYLNISPTDHYQFGGVATTNYYKNAVSYMLKRNKQAHFYIFSDDITWCKAEYKDLM oris F0302 [Prevotella PTFIECNKKNKSWRDMLLMSLCTNHINANSTFSWWGAWLSTKNGITICPTEFIHNVVTRDIYPETWVQL oris] Pseudo- WP_ 496239055 glycosyl 25.66 MIIVRLMGGMGNQLFQYATAFALSKRKSEPLVLDTRFFDHYTLHGGYKLDHFNISARILSKEEESLYPNQWA 130 gulbenkiania 496239055 transferase NLLLRYPIIDRAFKKWHVERQFTYQDRIYRMKRGQALLGYWQSELYFQEYRKEISAEFTLKEQSSVTAQQISV ferrooxidans family 11 AMQGGNSVAVHIRRGDYLSNPSALRTHGICLSGYYNHAMSLLNERINDAQFYIFSDDIAWAKENIKIGKTSK 2002; [Pseudo- NLIFIEGESVETDFWLMTQSKHHIIANSTFSWWGAWLANNTDEQLVICPSPWFDDKNLSETDLIPKSWIRLN Pseudo- gulbenkiania KDLPV gulbenkiania ferrooxidans] ferrooxidans Salmonella WP_ 446208786 protein 25.66 MYSCLSGGLGNQMFQYAAAYILKQYFQSTTLVLDDSYYYSQPKRDTVRSLELNQFNISYDRFSFTDEKEKIKLL 131 enterica 000286641.1 [Salmonella RKFKRNPFPKKISEILSIALFGKYALSDSAFYAVETIKNIDKACLFSFYQDADLLNKHKQLILPLFELRDDLLDICKN enterica] LDVYPLILRNNNTTALHIRRGDYLTNQHAAKYHGVLDTSYYNNAMEYVEREGKQNFIIFSDDVKWAQKAFL GNENCYIVNNGDYDYSAIDMYLMSLCKNNIIANSTYSWWGAWLNKSEDKLVISPKQWFLGNNETSLRNAS WIIL Carno- YP_ 554649642 glycosyl 25.59 MLIVKVYGGIGNQMFQYSFYKYLQKNNDDVFLDISDYKVHNHHNGFELIDVFNIEVKQADMSKFKGHVSSK 132 bacterium sp. 008718688.1 transferase NSIFYRLTSKLFKRNILGYSEFMDSNGISIVRNEKILTDHYFIGFWQDVLYLQSVEEEIKEAFNFKNVAIGKQNLE WN1359 family 11 LISLSESVESVSVHIRKGDYANNSDLSDICDLEYYEEAMKIIDSKVSEPLYFIFSDDIEWCKQKFGKRDNLIYVD [Carno- WNIAKKSYIDMLLMSKCKHNIIANSTFSWWGAWLNNNSKKIVICPKTWDRKKNENHLLLNDWIAI bacterium sp. WN1359] Prevotella WP_ 547227670 protein 25.58 MMKIIVNMACGLANRMFQYSYYLFLMHKGYNVKVDFYNSAKLAHEKVAWNDIFPKARIEQASFSDILKSG 133 sp. CAG:1185 021964668.1 [Prevotella GGSDVISKIRRKYLPFLSSVVNMPTAFDANLPVENKKLQYIIGVFQNANMVEAVEEDVKRCFKFQPFTDERNL sp. CAG:1185] KLQNEMQSCESVAIHVRKGKDYAQRIWYQNTCPIEYYQNAIRLISEKVNNPKLYVFTDNPEWVKEHFKDFPY TLVEGNPASGWGSHFDMQLMSVCKHNIISNSTYSWWSAFLNVHNEKIVIGPKVWFNPDSCSEFTSERILCK DWIAV Selenomonas WP_ 497331130 glycosyl- 25.58 MFQYAMASSVARRAGEILKLDLSWIRQMEKKLSADDIYGLGIFSFDEKFSTSNEVQKFLPSGKFSAKIYRAVN 134 sp. CM52 009645343.1 transferase, RRMPFSWRRVLEEGGMGWHPQIMEIRRSVYFYMGYWQSEKYFSDFIQEIFRKDFTFREEVRQSIEERRPIVE family 11 KIRKSDAVSLHIRRGDYAQNPALGEIFLSFTMQYYIDAARYISERVKTPVFFIFSDDIPWAKENLPLPYEVCYIDD [Selenomonas NIQTNEREIGHKSKGYEDMYLMTQCQHNIIANSSFSWWGAWLNHNPNKIVVAPKKWCNGSFNYADIVPE sp. CM52] QWVKL Bacteroides WP_ 494751213 protein 25.57 MEIVFIFNGLGNQMSQYALYSKRNLGCKVRYAYNIRSLSDHNGFELDRVFGITYPNNLFNKCINIIYRLLFAN 135 nordii; 007486621.1 [Bacteroides KYLFLVQKMIYMLRQMNVYSIKEKDNYDYDYKILTRHKGIVLYYGGWHSEKYFLSNADIIKDKFRFNISKLNSES Bacteroides nordii] LVLYHRLSSLNAVALHVRRGDYMAPEHYNVFGCVCGIEYYKAAIQYIQSQILNPVFIVFSNDIEWVKENITGIQ nordii MIFVDFNKKENSWMDMCLMSCCEHNIISNSTFSWWGAWLNNNKNKIVVCPKYFMSNIDTKDIYPESWIKI CL02T12C05 Para- WP_ 491855386 protein 25.54 MKKKDIILRVWGGVGNQLFIYAFAKVLSLITDCKVTLDIRTGFANDGYKRVYRLGDFSISLLPALRFTLLSFAQ 136 bacteroides 005635503.1 [Para- RKMPYIRHLLAYKFDFFEEDQKYPLETLDSFFKIYSDKNLYLQGYWQYFDFSSYRDVLLKDLRFEVEINNTYLYY merdae; bacteroides SDLIEKSNAVAIHFRRIQYEPVISIDYYKKAIKYISENVENPTFFIFSDDINWCRENLSINGICFFVENFKDELYELK Para- merdae] LMSQCNHFIIANSTFSWWGAWLSVNADKKVIMPDGYTDVSMNGSIVHI bacteroides merdae ATCC 43184; Para- bacteroides merdae CL09T00C40 Butyrivibrio WP_ 551024004 protein 25.51 MIIIQLKGGLGNQMFQYALYKELKHRGRDVDKIDDESGFIGDKLRVPVLDRFGVEYDRATKDEVIALTDSKMDI 137 sp. NC2007 022768139.1 [Butyribibrio FSRIRRKLTGRKTFRIDEMEGIFDPKILETENAYLVGYQWSEKYFTSPEVIEQIQEAFGKRPQEIMHDSVSWST sp. NC2007] LQQIECCESVSIHVRRTDYMDAEHIKIHNLCSEKYYKNAISKIREEHPNAVFFIFTDDKEWCKEHFKGPKFITVE LQEGEFTDVADMLLMSRCKHHIIANSSFSWWSAWLNDSPEKIVIAPSKWINNKKMDDIYTERMTKVAI Bacteroides WP_ 490430100 protein 25.5 MIVVYSNAGLANRMFHYALYKALEVKGIDVYFDEKSYVPEWSFETTTLMDVFPNIQYRESLQFKRASKKTFL 138 ovatus; 004302233.1 [Bacteroides DKIVIHCSNLFGGRYYVNYRFKYDDKLFTKLETNQDLCLIGLWQSEKYFMDVRQEIQKCFQYRSFVDDKNVKT Bacteroides ovatus] AQQMLSENSVAIHVRKGADYQQNRIWKNTCTIDYYRLAIDIRMHVQNPVFYVFTDNKDWVIENFTDLDY ovatus ATCC TLCDWNPTSGKQNYLDMQLMSCAKHNVIANSTYSWWGAWLNENSDKIVIAPKRWFNKIVTPDILPEQWI 8483; KI Bacteroides ovatus CL02T12C04 Mesotoga YP_ 389844033 glycosyl 25.5 MRVVWFGGGLGNQMFQYGLYCFLKKNNQEVKADCTQYSTTPMNNGFELERLFNLDIAHANLDVISKLTG 139 prima 006346113.1 transferase GNRLSPRKVIWKLFRKPKVYFEEKIPFSFDPDVLKGNNRYLKGYWQNMNYLEPCAKELRDVFTFPAFSSDNN MesG1.Ag.4.2; family protein KRLADEIAKVEAVGHVFRRGDFLKSSNLGLFGGICSDQYYLRAIQTMENTVVEPVFYVFCDDPQWAKNSFSD Mesotoga  [Mesotoga ARFTVIDWNIGSNSYRDMQLMSLCKHNIIANSTFSWWAAWLNRNPNRTVIAPERMVNRDLDFSGIFPND prima prima WIRLQG MesG1.Ag.4.2] Clostridium WP_ 545399562 glycosyl- 25.49 MIVLKLQGGLGNQMFEYAFARTIQEQKKDKKLILDTSDFQYDKQREYSLGHFILNENIEIDSSGKFNLWYDQR 140 sp. KLE 021639228.1 transferase, KNPLLKVGFKFWPKFQFQTLKFGIYVWDYAKYIPVDVSKKHKNILLHGLWQSDKYFSQISEIIRKEFAVKDEP 1755 family 11 SQGNKAWLERISSANAVCVHIRRGDFLAKGSVLLTCSNSYYLKAMEIISKKVNEPEFFIFSDDIEDVKKIFEFPG [Clostridium YQITLVNQSNPDYEELRLMSKCKHFIIANSTFSWWSSLLSENEDKVIVAPRLWYSDGRDTSALMRDEWIIIDN sp. KLE 1755] E Bacteroides WP_ 547321746 glycosyl- 25.42 MDLVTLSGGLGNQMFQFAFYWALKKRGKKVFLYKNKLAAKEHNGYETQTLFGVEEKCVDGLWMTRLLGC 141 plebeius 022052991.1 transferase, PLLGKILKHILFPHKIRERVLYNYSIYLPLFERNGLHWVGYWQSEKYFQDVADDIRRIFCFDHLSLNPATSAALK CAG:211 family 11 CMSEQVAVSVHIRRGDYYLPCNVATYGGLCTVEYYENAIRYVKEYPQAVFYVFSDDLDWVRENIPSAGKM [Bacteroides VFVDWNRGKDSWQDMFLMSKCHHNILANSSFSWWGAWLNTHPEKLVIAPERWANCPAPDALPDGWV plebeius RIEGVSRR CAG:211] Treponema WP_ 545448980 glycosyl- 25.4 MAIKIVKISGGLGNQMFCYAFACALQKCGHKVYVDTSLYRKATVHSGIDFCHNGLETERLFGIKFDEADTAD 142 lecithinoly- 021686002.1 transferase, VRRLSTSAEGLLNRIRRKYFTKKTHYIDTVFKYPELLSDKNDCYLEGYWQTEKYFLPIEKDIRRLFTFRPTLSEKS ticum; family 11 AAVQSALQAQQAAVLSASIHVRRGDFLNTKTLNVCTETYYNNAIKYAVKKHAVSRFIFSDDIPWCREHLCFC Treponema [Treponema NAHAVFIDWNTGNDSWQDMALMSMCRCNIIANSSFSWWAAWLNNASDKTVLAPAIWNRRQLEYVDRY lecithinoly- lecithinoly- YGYDYSDIVPESWIRIPID ticum ATCC ticum] 700332 Bacteroides WP_ 490419682 glycosyl- 25.34 MRLIKMTGGLGNQMFIYAFYLRMKKRHTNTRIDLSDMMHYNVHHGYEMHRVFNLPKTEFCINQPLKKVIE 143 eggerthii; 004291980.1 transferase FLFFKKIYERKQDPSSLLPFDKKYLWPLLYFKGFYQSWERFFADMENDIRIAFTFNSDLFNEKTQAMLTQIKHNE Bacteroides [Bacteroides HAVSLHIRRGDYLEPKHWKTTGSVCQLPYYLNAITEMNKRIEQPSYYVFSDDIAWVKENLPLPQAVFIDWNK eggerthii eggerthii] GAESWQDMMLMSHCRHHIICNSTFSWWGAWLNPRENKTVIMPERWFQHCDTPNIYPDGWIKVPVN DMS 20697 Bacteroides WP_ 491891563 glycosyl- 25.34 MRFIKMTGGLGNQMFIYAFYMRMKKHYSTRIDLSDMVHYKAHNGYEMHRVFNLPPIEFRINQPLKKVIEF 144 stercoris; 005656005.1 transferase LFFKKIYERKQVPSSLVPYDKKYFWPLLFKGFYQSERFFADMADDIRKAFTFNPRLSNRKTKEMSEQIDHDE Bacteroides [Bacteroides NAVSIHVRRGDYLEPKYWKTTGCVCQLPYYLNAIAEMNKRISQPSYYVFSDDIAWVKENPLPKAFFIDWNK stercoris stercoris] GAESWQDMMLMSRCRHHIICNSTFSWWGAWLNPRENKTVIMPERWFRHCETPDICPDKWIKVPINQPD ATCC 43183 SIQ Butyrivibrio YP_ 302671882 glycosyl 25.34 MIIIQLKGGMGNQMFQYALYRQLKKLGREKIDDETGFVDDELRIPVLQRFGISYDKATREEIVKLTDSKMDI 145 proteo- 003831842.1 transferase FSRIRRKLTGRKTFRIDEESGIFDPRILEVEDAYLVGYWQSDKYFANEEVEKEIREAFEKRPQEVMQDSVSWTI clasticus; 11  LQQIECCESVSLHIRRTDYIDEEHIHIHNICTEKYYKSAIDEVRNQYPSAVFFIFTDDKDWCRQHRFGPNFFVVD Butyrivibrio [Butyrivibrio LDEDTNTDIAEMTLMSRCKHHILANSSFSWWAAWLNDNPGKIVIAPSKWINNRKMDDIYTARMKKIAI proteo- proteo- clasticus clasticus B316 B316] Roseobacter WP_ 495504071 alpha-1,2- 25.34 MSPIVHFPSDRLLRYEHLSLWKTAMIYTRLLARLGNQMFQYAAGRGLAARLGVDFTDSRRAVHKGDGV 146 sp. GAI101 008228724.1 fucosyl- LTRVFDLDWAAPENMPPAQHERPLAYYAWRGLRRDPKIYRENGLGYNAAFETLPDNTYLGHYWQCERYFA transferase HIADDIRAAFVPRHPMSAWNADMARRIASGPSVSLHVRRGDYLTVGAHGICDQTYYDAALAAVMQGLPSP [Roseobacter TVYVFSDDPQWAKDNLPLTFEVVVDFNGPDSDYEDMRLMSLCQHNVIANSSFSWWGAWLNANPQKRV sp. GAI101] AGPANWFSNPKLSNPDILPSRWIRI Thalassobacter WP_ 544666256 alpha-1,2- 25.34 MGQDMIYSRIFGGLGNQLFQYATARAVSLRQGVELVLDTRLAPPGSHWAFGLDHFNISARIAEPSELPPSKD 147 arenae; 021099615.1 fucosyl- NFFKYVMWRAFGHDPAFMRERGLGYQSRIAQAPDGTYLHGYFQSERYFADVLDHLENELRIVTPPDTRNA Thalassobacter transferase EYADRIASAGHTVSLHVRRGDYVETSKSNSTHATCDEAYYLRALARLSEGKSDLKVFVFSDDPEWVRDNLKLP arenae [Thalasso- YDTTPVGHNGPDKPHEDLRLMSCCSDHVIANSTFSWWGGWLDRRPEARVVGPAKWFNNPKLVNPDILPE DSM 19593 bacter arenae] Prevotella WP_ 490511493 protein 25.33 MKIIKIIGGLGNQMFQYALAVALQKKWKDEEIKLDLHGFNGYHKHQGYQLDEIFGHRFKAASLKEVAQLAW 148 oris; 00437401.1 [Prevotella PYPHYQLWRVGSRLLPKRKTMVCESADCRFQSDLLNLEGSLYYDFYWQDERYFKAFRTEIIEAFKFTPLVGDS Prevotella oris] NRKVENMLKEGRFASLHVRRGDYLKEPLFQSTCDIAYYQRAISRLNQMADYPYCYLIFSNDIAWCKTHIEPLCD oris C735 GRRTHYVDWNHGKESYRDMQLMTFCKHHIIANSSFSWWGAWLSTANDGITIAPHQWYANDRKPSPAAE AWLKL Prevotella WP_ 490514606 protein 25.33 MKIVRIIGGLGNQMFQYALALALKQQQENEEVKLDLSAFRGYKKHGGFQLVQCFGTTLPAATWQEVAQLA 149 culorum; 004380180.1 [Prevotella WYYPHYQLWRLGHRVLPVCKTMLKEPDNGAFLPEVLQRKGDAYYEGCWQDERYFSHYRPAILQAFTFPTF Prevotella oulorum] TNPRNLAMQQQINTTESVAIHVRRGDYLHDALFRNTCGLAYFQRAITCILQHVAHPVFYVFSDDMAWCRQ oulorum HIQPLLQTNEAVFVDWNHGKASICDLHLMTLCRHHIIANSSFSWWGAWLSPHQAGWIIAPKQWYAHEEK F0390 MSPAAERWLKL Spirosoma WP_ 522084965 protein 25.33 MNRRVAVQLKGGLGNQLFQYALGRRLSLQLEAELLFDCSVLENRIPVTNFTFRSFDLDMFRIAGRVATPSDL 150 panaciterrae 020596174.1 [Spirosoma PLFPKSASIRSPWPHLVQLARLWKQGYSVYERGFAYNPKMLRQLSDRVYLNGYWQSYRYFEDIAATLRAD panaciterrae] CSFPDLPDSAVGLAGQINATNSICLHIRRTDFLQVPLHQVSNADYVGRAIAYMAERVNDPHFFVFSDDIAW CQTNLRLSYPVVFVPNELAGPKNSLHFRLMRYCKHFITANSTFSWWAWWLSEPSDGKVIVTPQTWFSDSRSI DDLIPANWIRL Butyrivibrio YP- 302669866 glycosyl- 25.26 MNYVEVKGGLGNQLFQYTFYKYLEKKSGHVLLHTDFFKNIDSFEEATKRKLGLDRFDCDFVAVSFFISCEKL 151 proteo- 003829826.1 transferase 11 VKESDYKDSMLSQDEVFYSGYWQNKRFFLEVMDDIRKDLLLKDENIQDEVKELAKELRAVDSVAIHRFFGDY clastiucs; [Butyrivibrio LSEQNKKIFTSLSVDYYQKAIAQLAERNGADLKGYIFTDEPEYVSGIIDQLGSIDIKLMPVREDYEDLYLMSCAR Butyrivibrio proteo- HHIIANSSFSWWGAALGDTESGITIAPAKWYVDGRTPDLYLRNSWISI proteo- clasticus clastiucs B316] B316 Butyrivibrio WP_ 551021623 protein 25.26 MIIIQLKGGLGNQMFQYALYKELKHRGREVKIDDVSGFVNDKLRVPVLDRFGVEYERATREEVVELTDSRMD 152 sp. XPD2006 022765786.1 [Butyrivibrio IFSRIRRKLTGRKTYRIDEMEGIFDPAILETENAYLVGYWQSEKYFTSPEVIEQIQEAFGKRPQEIMHDSVSWST sp. XPD2006] LQQIECCESVSIHVRRTDYVDAEHIKIHNLCSEKYYKNAIGKIREDHPNAVFFIFTDDKEWCKDHFKGPNFITVE LQEGEFTDVADMLLMSRCKHHIIANSSFSWWSAWLNDSPEKMVIAPSKWINNKKMDDIYTERMTRVAI Bacteroides WP_ 496041586 protein 25.24 MKIVNITGGLGNQMFQYAFAMALKYRNPQEEVFDIQHYNTIFFKKFKGINLHNGYEIDKVFPKAKLPVAGV 153 sp. 1_1_6 008766093.1 [Bacteroides RQLMKFSYWIPNYISLRLGRKFLPIRKKEYIPPSYMNYSYDEKALNWKGDGYFEGYWQSYNHFGDIKEELQK sp. 1_1_6] VYAHPKPNQYNAALISNLESCNSVGIHVRRGDYLAEFEFRGICGLDYYEKGIKEILSDEKKYVFFIFNDMQWC QENIAPLVGDNRIVFISGNKGKDSCWDMFLMTHCKDLIIANSSFSWWGAFLNKKVDRVICPKPWLNRDCNI DIYNPSWILCPCYSEDW Bacteroides YP_ 53713865 alpha-1,2- 25.17 MKIVTFQGGLGNQLFQYVFYWLDMRCDKDNIYGYYPKKGLRAHNGLEIEKVFEVKLPNSSLSTDLIVKSIKLI 154 fragilis; 099857.1 fucosyl- NKIFKNRQYISTDGRLDVNGVLFEGFWQDKYFWEDVDILNFRWPLKLDVTNSFIMTKIQANNSISIHIRRG Bacteroides transferase DYLLPKYRNIYGDICNEEYYQKAIEYILKCVDDPFFFVFSDDIDWAKSIINVSNVTFVNNNKGKDSYIDMFLMS fragilis [Bacteroides LCHHNIIANSTFSWWAAQLNKHSDKIMIAPIRWFKSLFKDPNIFTESWIRI YCH46 fragilis YCH46] Bacteroides WP_ 548260617 alpha-1,2- 25.17 MIKIVSFSGGLGNQLFQYLLVVYLRECGHQVYGYYNRKWLIGHNGLEVNNVFDIYLPKTNFIVNALVKVIRVL 155 sp. 9_1_42FAA 022477844.1 fucosyl- RCLGFKKYVATDTYNNPIAIFYDGYWQDQKYFNIIDSKLSFKKFDLSAENSILSKIKSNISVALHIRCGDYLSSS transferase NVEIYGGVCTKEYYEKALELVCKIKNVMFFVFSDDIEYAKLLLNLPNAIYVNANVGNSSFIDMYLMANCKVNV [Bacteroides IANSTFSYWAARLNQDNILTIYPKKWYNSKYAVPDIFPSEWVGV sp. 9_1_42FAA] [Coralio- WP_ 548260617 glycosyl- 25.17 MIIVKVQGGLGNQMFQYAFGRALSEKHSQDLYLDCSEYLRPSCKREYGLDHFNIRAKKASCDVKSMVTPH 156 margarita sp. 022477844.1 transferase FALRKKLKKIFAVPYSLSPTHILERNFNFQPSILEFNCGYFDGFWQTQKYFSGISDIVRKDTFKDAVKYSGGET CAG:312 family 11 FAKITSLNSVSLHIRRGDYYKVKRTRKRFSVIRAGYFKRAVEYMRSKLDTPHFFIFTDDPKWVSENFPAGEDYT [Coralio- LVSSSGMYEDLFLMAQCRHNIIFNSSFSWWGAWLNGNPGKIVVAPDMWFTPHYKLDYSDVVPEEWIKLN margarita sp. TGYFESKEF CAG:312] Pseudor- WP_ 550957292 alpha-1,2- 25.17 MIVMQIKGGLGNQMFQYAAGRALSLQTGMPLHLDLRYYRREREHGYGLGAFNIEASPLDESLLPPLPRESPL 157 hodobacter 022705649.1 fucosyl- AWLIWRLGRRGPNLVRENGMGFNPTLSNVTKPAWITGYFQSERYFAAHAATIRAELTPVAAPDLVNARWL gerrugineus transferase AEIAAEPRAVSLHVRRGDYVRDAKAAAKHGSCTPAYYERALAHITARMGTAPVVYAFSDDPAWVRENLRLP [Pseudor- AEIRVPGHNDTAGNVEDLRLMSACRHHIVANSSFSWWGAWLNPRADKIVASPARWFADPAFTNPDIWPE hodobacter AWARIEG ferrugineus] Escherichia YP_ 215487252 fucosyl- 25.16 MMYCCLSGGLGNQMFQYAAAYILKQHFPDTILVLDDSYYFNQPQKDTIRHLELDQFKIIFDRFSSKDEKVKIN 158 coli; 002329683.1 transferase RLRKHKKIPLLNSFLQFTAIKLCNKYLSNDSAYYNPESIKNIDVACLFSFYQDSKLLNEHRDLILPLFEIRDDLRVL Escherichia [Escherichia CHNLQIYSLITDSKNITSIHVRRGDYVNNKHAAKFHGTLSMDYYISAMEYIESECCGSQTFIIFTDDVIWAKEKFS coli O127:H6 coli O127:H6 KYSNCLVADADENKFSVIDMYLMSLCNNNIIANSTYSWWGAWLNRSEDKLVIAPKQWYISGNECKLKNEN str. E2348/69 str. E2348/69 WIAM Lachno- WP_ 511537894 protein 25.16 MIIIKVMGGLGNQMQQYALYEKFKSIGKNKVKLDISWFEDSSVQEKVFARRSLELRQFKDLQFDTCSAEEKEA 159 spiraceae 016359991.1 [Lachno- LLGKSGILGKLERKLIPARNKHFYESDIYHSEVFNMSDAYLEGHWACEKYYHDIMPLLQEKIQFPESANSQNIT bacterium spiraceae VKKRMKAENSVSIHIRRGDYLDPENEAMFGGICTNSYYKAAEEYIKSRVPDTHFYLFSDDTAYLRENYHGDEY 3_1_57FAA_ bacterium TIVDWNKGEDSFYDMELMSCCRHNICANSTFSFWGARLNRTPDKIVIPAKHKNSQEIEPQLLHELWDNW CT1 3_1_57FAA_ VIIDGDGRIV CT1] Butyrivibrio WP_ 551010878 glycosyl- 25.09 MKPLVSLIVPVLNVEKYLEQCLTSISSQTYDNFEVILVVGKCIDNSENICKKWCEKDHRFRIEPQLKSCLGYARN 160 fibrisolvens 022755397.1 transferase VGIDAAKGEYIAFCDSDDCITSDFLSCFVDTALKNSSDIVETQFTLCDQNLSPIYDYDRNILGHILGHGFLEYTSA [Butyrivibrio PSVWKYFVKRDIFTSNNLHYPEIRFGEDISMYSLLFSYCNKIDYVEKPTYLYRQVPSSLMNNPQGKRKRYESLF fibrisolvens] DIIDFVTNEFKTRLLFQKSWLKLLFQLEMHSASIISDSATSDDEAISMRQEISGYLKKVFPVKNTIFEVTALGWG GEIVSSIASKFNTLHGVSSSNMFNFYFFELLEDSTRKKLEEMIINFSPDIFLIDLISEADYLSSYKGNLGTFVKNW KIGFSIGMKMIQTHSNNSSIFLLENYMQQAPDHVDNTNEILKMLYDDIKINHPDIICISPAPDILNRSSEPELPCI YQLKLVSDKLHTMYSPVINCVETKGGLGNQIFQYVSKYIEKMTGYRPLLHIGFFDYVKAIPGGTKRIFSLDKLF PDIETTSGKIPCSHVVEEKSFISNPGSDIFYRGYWQDIRYFSDVKDEVLESFNVDTSSMSKDVIDFADTIRNANS IAMHIRRQDYLNENNVSLFEQLSIDYYKSAVDMIRKEYADDLVLFIFSDDPEYANSIADSFDIEGFVMPLHKDY EDLYLITLAHHHIIANSTFSLWGALLSARKDGIRIAPRNWFKGTPATNLYPDKWLIL Anaeromusa WP_ 517532751 protein 25.08 MFCVRIYGGLGNQMFQYALGRAMAKHYSETAAFDLSWYEQKIKPGFEASVCQYNIELSRKDRPKAWYEPIL 161 acida- 018702959.1 [Anaeromusa KRISRHTDKLEMWFGLFFFEKKYHTDSTVFERGLCKKNITLDGYWQSYKYFSAIEDDLRRELTIPKEREELIAISRS minophila acida- LPENSVSIHVRRGDYVSNPKANAMHGTCSWEYYQAAIEKMTGLVKEPQYWFSDDITWTKENLPLPNAMYI minophila] GRELGLFDYEELILMSRCKHNIMANSTFSWWGASLNSNPNKVVIAPRKWFRHKKIKVNDLFPSSWVVL Bacteroides WP_ 496044479 glycosyl 25.08 MDIVVIFNGLGNQMSQYAFYLAKKKDNLNCHVIFDPKSTNVHNGAELKRVFGIELNRNYLDKIISYFYGYIFN 162 sp. 2_1_16 008768986.1 transferase KRIVNKLFSLVGIRMIYEPKNYDYFEELLKPSSNFISFYWGGWHSEKYFKDIELEVKKVFKFPEVTNSPYFTEWF family 11 NKIFLDNNSVSIHIRRGDYLDKPSDPYYQFNGVCTIDYYEKAILYLKERILEPNFYIFSNDINWCMKTFGTENMY [Bacteroides YVDCNKGKDSWRDMYLMSECRHHINANSTFSWWAAWLSPYSNGIVLHPKYFIKDIETKDYYPQKWIMIE sp. 2_1_16] Chlorobium YP_ 189500849 glycosyl 25.08 MDKVVVHLTGGLGNQMFQYALGRSISINRNCPLLLNTSFYDTYDKFSCGLSRYNVKAEFIKKNSYYNNKYYRY 163 phaeo- 001960319.1 transferase VIRLLSRYGVACYFGSYYEKKIFSYDEKVYKRSCVSYYGTWQSYGYFDSIRDILLRDYEMVGCLEEEVEKYVSDIK bacteroides; family protein RVDSVSLHIRRGDYFDNKRLQSIHGILTMEYYYKAMSLFPDSSVFYVFSDDIEWVRENLITNTNIVYVVLESDN Chlorobium [Chlorobium PENEIYLMSLCKNNIISNSTFSWWGAWLNKNKYKKVIAPRMWYKDNQSSSDLMPSDWCLI phaeo- phaeo- bacteroides bacteroides BS1 BS1] Treponema WP_ 551312724 protein 25.08 MIVISMGGGLGNQMFEYAFYTQLKHLYPKSEIKVDTKYAFPYSHNGIEVFKIFGLNPPEANWKEVHSLVKTYP 164 bryantii 022932606.1 [Treponema IEGNKAHFIKFFLYRILRKANLVEREPTSFCKQKDFTEFYNSFFELPQNNKSFYLYGPFVNYNYFAAIHNEIMDLYT bryantii] FPEITDVTNIEYKRKIESSHSISIHIRRGDYITEGVPLVPDAYYREALVYINKKIEDPHFFVFTDDKDYCKSLFSDN QNFTIVEGNTGANSFRDMQLMSLCKHNIIANSTFSFWGAFLNKNSEKIVIAPNIAFKDCSCPYICPDWIIL Bacteroides YP_ 675358171 LPS 25 MVIAKLFGGLGNQMFIYAAAKGIAQISNQKLTFDIYTGFEDDSRFRRVYELKQFNLSVQESRRWMSFRYPLG 165 fragilis; 005110943.1 biosynthesis RILRKISRKIGFCIPLVNFKFIVEKKPYHFQNEIMRIASFSSIYLEGYFQSYKYFSKIEAQIREDFKFTKEVIGSVEK Bacteroides alpha-1,2- ASFITNSRYTPVAIGVRRYSEMKGEFGELAVVEHDYYDAAIKYIANKVPNLIFIVFSEDIDWVKKNLKLDYPVYF fragilis fucosyl- VTSKKGELAAIQDMYLMSLCNHHIISNSSFYWWGAYLASTNNHIVIAPSVFLNKDCTPIDWVII 638R transferase [Bacteroides fragilis 638R] Firmicutes WP_ 547951298 protein 25 MSGGLGNQMFYALYMKLTAMGREVKFDDINEYRGEKAWPIMLAVFGIEYPRATWDEIVAFTDGSMDFS 166 bacterium 022352105.1 [Firmicutes KRLKRLFRGRHPIEYVEQGFYDPKVLSFENMYLKGSFQSQRYFEDILEEVQETFRFPELKDMNLPAPLYETTEK CAG:534 bacterium YLLRIEGCNAVGLHMYRGDSRSNEELYDGICTEKYYEGAVRFIQDKCPDAKFFIFSNEPKWVKGWVISLMKS CAG:534] QIREDMSREEIRALEDHFVLIENNTEYTGYLDMFLMSRCRHNIISNSSFSWWAFINENPDKLVTAPSRWVN GVPSEDVYVKGMTLIDEKGRVERTIKE Firmicutes WP_ 547971670 glycosyl- 25 MVIVKIGDGLGNQMFNYVCGYSVAKHDNDTLLLDTSDVDNSTLRTYDLDKFNIDFTDRESFTNKGFFHKVYK 167 bacterium 022368748.1 transferase RLRRSLKYNVIYESRTENCPCVLDVYRRKFIRDGYLHGYFQNLCYFKTCKEDIMRQFTPKEPFSAKADELIHRFA CAG:882 family 11 TENTCSVHVRGGDIKPLSIKYYKDALDKIGEAKKDMRFIVFSNVRNLAEEYIKELGVDAEFIWDLGEFTDIEELF [Firmicutes LMKACRRHILSDSTFSRWAALLDEKSEEVFVPFSPDADKIYMPEWIMEEYDGNEEKR bacterium CAG:882] Vibrio WP_ 491639353 glycosyl- 25 MVIVKVSGGLGNQLFQYAIGCAISNRLSCELLLDTSFYPKQSLRKYELDKFNIKAKVATQKEVFSCGGGDDLLS 168 parahae- 005496882.1 transferase RFLRKLNLSSLFFPNYIKEKESLVYLAEISHCKSGSFLDGYWQNPQYFSDIKDELVKQIVPIMPLSSPALEWQNII molyticus; family 11 INTKNCVSLHVRRGDYVNNAHTNSVHGVCDLSYYREAITNIHETVEKPKFFVFSDDISWCKDNLGSLGHFTYV Vibrio [Vibrio DNTLSAIDDLMLMSFCEHHIIANSTFSWWGAWLNDHGITIAPKRWFSSVERNNKDLFPEKWLIL parahae- parahae- molyticus molyticus] 10329; Vibrio parahae- molyticus; 10296; Vibrio parahae- molyticus; 12310; Vibrio parahae- molyticus; 10290 Herba- WP_ 493509348 glycosyl- 24.92 MIVSRLIGGLGNQMFQYAAGRALALRRGVPFAIDSRAFADYKTHAFGMQCFCADQTEAPSRLLPNPPAEGR 169 spirillum 006463714.1 transferase LQRLLRRFLPNPLRVYTEKTFTFDEAVLSLPDGIYLDGYWQSEKYFADFADDIRKDFAVKAAPSAPNQAWLEL frisingense; family protein IGRTHSVSLHIRRGDYVSNAAAAANHGTCDLGYYERAVAHLHQVTGQAPELFVFSDDLDWVATNLQLPYT Herba- [Herba- MHLVRDNDAATNFEDLRLMTACRHHIVANSSFSWWGAWLDGRSESITIAPARWFVADTPDARDLVPQR spirillum spirillum WVRL frisingense; frisingense] GSF30 Rhizobium WP_ 495034125 Glycosyl- 24.92 MIITRILGGLGNQMFQYAAGRALAIANEAELKLDLIEMGAYKLRPFALDQFNIKAAIAQPDEVPAKPKRGLLR 170 sp. CF080 007759661.1 transferase KFTSAFKPDRSSCERIVENGLTFDSRVPALRGSLHLSGYWQSEQYFASSADAIRSDFSLKSPLGPARQDVLARI family 11 GAATTPVSIHVRRGDYVTNPSANAVHGTCEPPWYHEAMRRMLDRAGDASFFVFSDEPQWARDNLQSSRP [Rhizobium MVFIEPQNNGRDGEDMHLMAACHAHIIANSSFSWWGAWLNPRPNKHVIAPRQWFRAPDKDDRDIVPA sp. CF080] TWERL Verrucomicro- WP_ 497645196 glycosyl- 24.85 MVISHISEGLGNQMFQYAAGRRLSYHLGTTLKLDDYHYRLHPFRSFQLDRFLITSPIATDAEISHLCPLEGLAR 171 bium 009959380.1 transferase AIRARLPGKLRGATLRLLGNLGLGSPYQPRLHSFKEETPKQPLLIGKVVSERHFHFDPDVLECPDNVCLVGYW spinosum family 11 QDERYFGEIRDILLRELTLKSPPAGATKAVLERIQRSSSVSLHVRRGDKTKSSSYHCTSLEYCLAAMSEMRARL [Verrucomicro- QAPTFFVFSDDWDWVREQIPCSSSVIHVDHNRAEDVSEDFRLMKSCDHHIIASSSLSWWAAWLGTNENSF bium VSPPADRWLNFSNHFTADVLPPHWIQLDGSSLLPAQ spinosum] Fibrella YP_ 436833833 glycosyl- 24.83 MTANRVLVNSPMVIAKITSGLGNQLFQYALGRHLALQGNTSLWFDLRFHQEYATDTPRKFKLDRFNVRYN 172 aestuarina; 007319049.1 transferase LLDSSPWLYASKATRLLPGRSLRPLIDTRFEADFHFDPTVIRPAAPLTILWGFWQSEDKYFAQSTPQIRQELTFN Fibrella family 11 RPLSDTFVGYQQQIEQAEVPISVHVRRGDYVTHPEFSQSFGFVGLAYYQKALAHLQDLFPNATLFFFSDDPD aestuarina [Fibrella WVRANIVTEQPHVFVQNSGPDADVDDLQLMSLCHHHVIANSSFSWWGAWLNPRPDKVVIGPQRWFAN BUZ 2 aestuarina KPWDTKDLLPSGWLRL BUZ 2] Rhodobacter WP_ 563380195 alpha-1,2- 24.83 MIHMRLVGGLGNQLFQYACGRAVALRHGETLVLDTRELSRGAAHAVFGLDHFAIRARMGASADLPPPRSR 173 sp. CACIA14H1 023665745.1 fucosyl- VLAYGLWRAGFMAPRFLRERGLGVNPAVLAAGDGTYLHGYFQSEAYFRDVVPQIRPELEIVTPPSDDNLRW transferase ASRIAGDDRAVSLHVRRGDYVASAKGQQVHGTCDADYYARAVAAIRARAGIDPRLYVFSDDPHWARDNLA [Rhodobacter LDAETVVLDHNPPGAAVEDMRLMGVCRHHIIANSSFSWWGAWRNPSAGKVVVAPVRWFADPKLHNPDI sp. CACIA14H1] CPPEWLRV Rhodo- NP_ 32475785 fucosyl- 24.83 MATSAHLHLSDEKQTLDSKASDRDCATTEASASDKTCTISISGGLGNQMLQYAAGRALSIHHDCLSQLDLKF 174 pirellula 868779.1 transferase YSSKRHRSYELDAFPIQAHRSIKPSFFSQILSKIQSESKHVPTYQEQSKRFDPAFFNTEPPVKIRGYFFSEKYFSPY baltica SH 1; [Rhodo- ADQIRTELTPPIPPDQPADRMAIRLKECVSTSLHVRRGDYVTNANARQRFWCCTSEYFEAAIERLPTDSTVFV Rhodo- pirellula FSDDIEWAKQNIRSSRTTVYVNDELKKAGSPETGLRDLWLMTHAKSHIIANSSFSWWGAWLANSEANLTIA pirellula baltica SH 1] PKKWFNDPEIDDSDIVPSSWHRI baltica Spirosoma WP_ 522092845 protein 24.76 MVVVELMGGLGNQMFQYAFGMQLAHQRQDTLTVSTFLLSNKLLANLRNYTYRPFELCIFGIDKPKASPFNL 175 spitsbergense 020604054.1 [Spirosoma LRALLPFDLNTSLLRETDDPEAVIPAASARIVCVGYWQSEHYFEEVTVHVREKFIFRQPFNSFTSRLANNLNGI spitsbergense] PNSVFVHIRRGDYVTNKGANAHHGLCDRTYYERAVTFMREHLENPLFFIFSDDLEWVSQELGPILEPATYVG GNQKNDSWQDMYLMSLCRHAIVANSSFSWWGAWLSPHASKIVVAPKEWFGKPLLPVKTNDLIPSNWIRI uncultured EKD71402.1 406938106 protein 24.76 MNAIIPRITGGIGNQLFIYAAARRMAIANSMNLVIDDTSGFKYDVLYKRFYQLEKFNITSRMATPTERLEPFSK 176 bacterium ACD_ IRRYLKRKINKTYPFAQRAYITQEKSGFDPRLLVFRPKGNVYLDGYWQSENYFKDIEGIIRQDLIIKSPSDSLNIA 46C00193 TAERIKNTLAIAVHVRFFDMVDISDSSNCQSNYYHTAIAKMEEKIPNAHYFIFSDKPVLARLAMPLPDDRITIID G0003 HNIGDMNAYADLWLMSLCKHFVIANSTFSWWGAWLSDNKEKIVIAPDITITSGVTQWGFDGLIPDEWIKL [uncultured bacterium] Prevotella WP_ 494008437 protein 24.75 MDVIVIFNGLGNQMSQYAFYLEKRLRNRQTTYFVLNPRSTYELERLFGIPYRSNLMCRMIYKLLDKAYFSNHI 177 micans; 006950883.1 [Prevotella RLKKILRTALNAVGIRLIVEPITRNYSLSNFTHHPGLTFYRGGWHSELNFTSVVTELRRKFIFPPSDDEEFKRISAL Prevotella micans] IIRTQSISLHIRRGDYLDYSEYQGVCTEEYYERAIEYIRSHVENPVFFVSDDKEYAINKFSGDDSFRIVDFNTGE micans F0438 NSWRDMQLMSLCRHHILANSTFSWWGAWLDSAPEKIVLHPIYHMRDVPTRDFYPHNWIGISGE Thermo- AHB87954.1 564737556 alpha-1,2- 24.75 MIIVRLYGGLGNQMFQYAAGLALSLRHAVPLRFDLDWFDGVRLHQGLELHRVFDLDLPRAAPSEMRQVLG 178 synechococcus fucosyl- SFSHPLVRRLLVRRRLRWLLPQGYALEPHFHYWPGFEALGPKAYLDGYWQSERYFSEYQDAVRAAFRFAQP sp. NK55 transferase LDERNRQIVEEMAACESVSLHVRRGDFVQDPVVRRVHGVDLSAYYPRAVALLMERMREPRFYVFSDDPD [Thermo- WVRANLKLPAPMIVIDHNRGEHSFRDMQLMSACRHHILANSSFSWWGAWLNSQPHKLVIAPKRWFNVD synechococcus DFDTRDLYCSGWTVL sp. NK55] Coleo- WP_ 493031416 Glycosyl- 24.73 MLSLNKNFLFVHIPKSCILKEVYIYMISFPNLGKGVRLGNQMFQYAFLRSTARRLGVKFYCPAWSGDSLFTLN 179 fasciculus 006100814.1 transferase DQEERVSQPEGITKQYRQGLNPGFSENALSIQDGTEISGYFQSDKYYDNPDLVRQWFSLKEEKIASIRDRFSRL chthono- family 11 NFANSVGMHLRFGDVVGQLKRPPMRRSYYKKALSYIPNQELILVFSDEPERTKKMLDGLSGNFLFLSGHKNY plastes; [Coleo- EDLYLMTKCQHFICSYSTFSWWGAWLGGERERTVIYPKEGQYRPGYGRKAEGVSCESWIEVQSLRGFLDDY Coleo- fasciculus RLVSRLEKRLPKSLMNFFY fasciculus chthono- chthono- plastes] plastes PCC 7420 Bacteroides WP_ 517496220 glycosyl- 24.66 MRLIKMTGGLGNQMFIYAFYLRMKKRHTNTRIDLSDMMHYNVHHGYEMHHVFNLPKTEFCINQPLKKVIE 180 gallinarum 018666797.1 transferase FLFFKKIYERKQDSSNLLPFDKKYFWPLLYFKGFYQSERFFADMENDIRKAFTFNSGLFNEKTQTMLKQIEHNE [Bacteroides HAVSLHVRRGDYLEPKHWKTTGSVCQLPYYINAIAEMNRRIEQPFYYVFSDDIAWVKENLPLPQAVFIDWN gallinarum] KGVESWQDMMLMSHCRHHIICNSTFTWWGAWLNPKENKTVIMPERFQHCETPNIYPAGWIKVPIN Firmicutes WP_ 547967507 glycosyl- 24.66 MNNVEIMGGLGKQLFQYAFSRYLQKLGVKNVVLRKDFFTIQFPENNGITKREVFLDKYNTRYVAAAGEKTYR 181 bacterium 022367483.1 transferase DYCDENDYRDDYAIGSDEVLYEGYWQNIDFYNVVRKEMQEELKLKPEFIDNSMAAVEKDMSSCNSVALHIR CAG:882 GT11 family RSDYLTQVNAQIFEQLTQDYYASAVSIIEQYTHEKPVLYIFSDDPEYAAENMKDFMGCRTVIMPPCEPYQDM [Firmicutes YLMTRAKHNIIANSTFSWWGATLNANPDNITVAPSRWMKGRTVNLYHKDWITL bacterium CAG:882] Bacteroides WP_ 494296741 protein 24.6 MIAVNVAGLANQMFHYAFGFGLMAKGLDVCFDQSNFKPRSQWAFELVRLQDAFPSIDIKVMPEGHFK 182 xylanisolvens; 008021494.1 [Bacteroides WVFPSLPRNGLERRFQEFMKKWHNFIGDEVYIDEPMYGYVPDMEKCATRNCIYKGFWQSEKYFRHCEDDI Bacteroides xylanisolvens] RKTFTFPLFDELKNIEVAAKMSQENSVAIHLRKGDDYMQSELMGKGLCTVDYYMKAIDYMRKHINNPHFY xylanisolvens VFTDNPCWVKDNLPEFEYILVDWNEVSGKRNFRDMQLMSCAKHNIIGNSTYSWWAWLNANQDKIVVG CL03T12C04 PKRFFNPINSFFSTCDIMCEDWISL Geobacter YP_ 322418503 glycosyl- 24.58 MIGMVIFRAYNGLGNQMFQYALGRHLALLNEAELKIDTTAFADDPLREYELHRLKVQGSIATPDEIAFFREM 183 sp. M18 004197726.1 transferase ENTHPQAYLRLTQKSRLFDPAILSARGNIYLHGFWQTEKYFADIREILLDEFEPIVPAGEDSIKVLSHMKATNA family protein VALHVRRSDYVSNPMTLRHHGVLPLDYYREAVRRIAGMVPDPVFFIFSDDPQWAKDNIRLEYPAFCVDAHD [Geobacter ASNGHEDLRLMRNCKHFIIANSSFSWWGAWLSQNTGKKVVAPLKWFAKPEIDTRDIVPLQWIRI sp. M18] Ruegeria YP_ 56698215 alpha-1,2- 24.57 MITTRLHGRLGNQMFQYAAARGLAARLGTQVALDTRLAESRGEGVLTRVFDLDLAQPDQLPPLKGDGLLR 184 pomeroyi 168587.1 fucosyl- HGAWRLLGLAPRFRREHGLGYNAAIETWDDGTYLHGYWQSERYFAHIAARIRADFAFPAFSNSQNAEMAA DSS-3 transferase RIGDTDAISLHVRRGDYVALAAHTLCDQRYYAAALTRLLEGVAGDPVVYLFSDDPAWARDNLALPVQKVVV [Ruegeria DFNGPETDFEDMRLMSLCRHNIIGNSSFSWWAAWLNAHPGKRIAAPASWFGDAKLHNPDLLPPDWLKIE pomeroyi V DSS-3] Lachno- WP_ 511037973 protein 24.52 MIIIQLAGGLGNQMQQYAMYQKLLSLGKKVKLDISWFEEKNRQKNVYARRELELNYFKKAEYEACTEEERKA 185 spiraceae 016291997.1 [Lachno- LVGEGGFAGKIKGKLFPGTRKIFRETEMYHPEIFDFEDRYLYGYFACEKYYADIMEILQEQFVFPPSGNPENQK bacterium spiraceae MAERIADGESVSLHIRRGDYLDAENMAMFGNICTEEYYAGAIREMKKIYPSAHFFVFSDDIPYAKETYSGEEF 28-4 bacterium TVVDINRGKDSFFDIWLMSGCRHNICANSTFSFWGARLNRNKGKVVMRPFIHKNSQKFEPELMHELWKG 28-4] WVFIDNRGNIC Prevotella WP_ 547254188 glycosyl- 24.49 MRILVFTGGLGNQMFEYAFYKHLKSCFPKESFYGHYGVKLKEHYGLEINKWFDVTLPPAKWWTLPPVVGLFYL 186 sp. CAG:1092 021989703.1 transferase YKKLVPNSKWLDLFQREWKHKDAKVFFFPFKFTKQYFPKENGWLKWKVDEASLCEKNKKLLQVIHDEETDCFV family 11 HVRRGDYLASNFKSIFEGCCTLDYYKRALEYMNKNNPKVRFICFSDDLEWMRKNLPMDDSAIYVDWNTGT [Prevotella DSPLDMYMMSQCDNGIIANSSFSYWGAYLGGKKTTVIYPQKWWNMEGGNPNIFMDEWLGM sp. CAG:1092] Spirosoma WP_ 517447743 protein 24.41 MVISVLSGGLGNQLFQYAFGLKLAALQLQTELRLERHLLESKAIARLRQYTPRTYELDTFGVEAPAASLMDTVS 187 luteum 018618567.1 [Spirosoma CLSRVASLDKTALLLRESTLTPNAINNLNNRVRDVVCLGYWQSEEYFRPATEQLRKHLVFRKNPAQSRSMAD luteum] TILSCQNAAFVHIRRGDYVTNTHANQHHGLCDVSYYRRACEYVKECIPDVQFFVFSDDPDWAKRELGIHLQP ARFIDHNRGADSWQDMYLMSLCRHAIVANSSFSWWGAWLNPVAERLVVAPGQWFVNQPVLSQQIIPPH WHCL Marinomonas YP_ 333906886 glycosyl- 24.34 MIIVDLSGGLGNQMFQYACARSLSIELNLPLKVVYGSLASQTVHNGYELNRVFGLDLEFATENDMQKNLGFF 188 posidonica 004480472.1 transferase LSKPILRKIFSKKPLNNLKFQNFFPENSFNYSSLFSYIKDSGFLQGYWQTEKYFLNHKSQILKDFCFVNMDDE IVIA-Po- family protein TNISIANDIQSGHSISIHVRRGDYLTNLKAKAIHGHCSLDYYLKAIEFLQEKIGESRLFIFSDDPEWVSENIATRFS 181; [Marinomonas DVSVIQHNRGVKSFNDMRLMSMCDHHIIANSSFSWWGAWLNPSQNKKIIAPKNWFVTDKMNTIDLIPSS Marinomonas posidonica WILK posidonica IVIA-Po-181] Bacteroides; WP_ 492425792 glycosyl- 24.32 MKIVVFKGGLGNQLFQYAFYKYLSRKDETFYFYNDAWYNVSHNGFELDKYFKTDDLKKCSRFWIILFKTILSKI 189 Bacteroides 005839979.1 transferase YHWKIYVVGSVEYQYPNHLFQAGYFLDKKYYDENTIDFKHLLLSEKNQSLLKDIQNSNSVGVHIRRGDYMTK sp. 4_3_47FAA; family 11 QNLVIFGNICTQKYYHDAIRIITEKVNDAVFYVFSDDISWVQTHLDIPNAVYVNWNTGESSIYDMYLMSSCKY Bacteroides [Bacteroides NIIANSTFSYWAARLNKKTNMVIYPSKWYNTFTPDIFPESWCGI sp. 3_1_40A; Bacteroides dorei 5_1_36/D4; Bacteroides vulgatus PC510; Bacteroides dorei CL03T12C01; Bacteroides vulgatus dnLKV7 Candidatus WP_ 519013556 protein 24.32 MTIRIKLTGGLGNQMFQFATGFAIAKKKNVRSLSDLKYINKRKLFNGFELQKIFNIYSKVSFLNKTLSFKSINFTE 190 Pelagibacter 020169431.1 [Candidatus ILNRIDTTFYNFKEPHFHYTSNILNLPKHSFLDGYWQSELYFNEFATEIKRIFNFSGKLDKSVLLVADDINRNNSI ubique Pelagibacter SIHIRRGDFLLKQNNNHHTDLKEYYLKAINETSKIFKNPKYFIFSDDTSWTVDNFVIDHPYIIVDINFGARSFLD ubique] MYLMSLCKSNIIANSSFSWWSAWLNNNKDKIIYAPKNWFNDKSICTDDLIPESWNIIL Bacteroides WP_ 519013556 protein 24.29 MSVIINMACGLANRMFQYAFYLYLQKEGYDAYVDYFTRADLVHENVDWLRIFPEATFRRATARDIRKMGG 191 sp. CAG:875 020169431.1 [Bacteroides GHDCFSRLRRKLLPMTTKVLETSGAFEIILPPKNRDSYLLGAFQSAKMVESVDAEVRRIFTFPEFESGKNQYFQ sp. CAG:875] TRLAQENSVGLHIRKGKDYQERIWYKNTCGVEYYRKAVDLMKEKVDSPSFYVFTDNPAWVKENLSWLEYKL VDGNPGSGWGSHCDMQLMSLCKHNIISNSSYSWWGAYLNNTLNKIVVCPRIWFNPESTKDFSSNPLLAEG WISL Butyrivibrio WP_ 551011911 protein 24.29 MIIIKLQGGLGNQLFLYGLYKNLKHLKRDVKMDIESGFEGDELRKPCLDCMNLEYAIATRDEVTDIRDSYMDI 192 fibrisolvens 022756327.1 [Butyrivibrio FSRIRRKITGRKTFDYYEPEDGNYDPKVLEMTKAYLNGYFQSEKYFGDEESVKALKDELTKGKEDILTSTDLITKI fibrisolvens] YHDIKNSESVSLHIRRGDYLTPGIIETYGGICTDEYYDKAIAMIRETFPEARFFIFSNDIEWCKEKFAGDKNILFV NTIGINLDSEDNIKIGKSDKDISEYRDLAELYLMSACKHHILANSSFSWWGAWLSDHEGMTIAPSKWLNNKN MTDIYTKDMLLI Roseburia YP_ 347532692 glycosyl- 24.22 MVTVKIGDGMGNQMYNYACGYAAAKRSGEKLRLDISECDNSTLRDYELDHFRVVYDEKESFPNRTFWQKL 193 hominis; 004839455.1 transferase YKRLRRDIRYHVIRERDMYAVDARVFVPARRGRYLGHYWQCLGYFEEYLDDLREMFTPAYEQTDAVRELM Roseburia family protein QQFTQTPTCALHVRGGDLGGPNRAYFQQAIARMQKEKPDVTFIVFTNDPKAKECLDDGEARMYRIAEFGE hominis A2- [Roseburia ALSDIDEFFLMSACQNQIISNSTYSTWAAYLNTLPGRIVIGPKFHGVEQMALPDWIVLDGGACQKGEIDAV 183 hominis A2- 183] Rhodo- WP_ 495934621 alpha-1,2- 24.16 MATSVHPHLSDGKQALDSKAAQQVCSTQAASASDRACTISIGGLGNQMLQYAAGRALSIHHDCPLQLDLK 194 pirellula; 008659200.1 fucosyl- FYSSKRHRSYELDAFPIQAQRWIKPSFFSQVLDKIQGESKSAPTYEEQSKRFDRAFFDIELPARIRGYFFSEKYFL Rhodo- transferase PYADQIRTELTPPVPLDQPARDMAQRLSEGMSTSLHVRRGDYVSNANARQRFWSCTSEYFEAAIEQMPAD pirellula [Rhodo- STVFVFSDDIEWAKQNIRSSRPTVYVNDELKLAGSPETGLRDLWLMTHASKSHIIANSSFSWWGAWLSGSEA europaea 6C pirellula NLTIAPKKWFNDPEIDDSDIVPTSWRRI europaea] Rudanelia WP_ 518832653 proein 24.16 MVIAKITSGLGNQLFQYALGRHLAIQNQTRLWFDLRYYHRTYETDTPRQFKLDRFSIDYDLLDYSPWLYVSKA 195 lutea 019988573.1 [Rudanelia TRLLPGRSLRPLFDTRKEPHFHLDPAVPNAKGAFITLDGFWQSEGYFASNAATIRRELTFTRQPGPMYARYR lutea] QQIEQTQTPVSVHIRRGDYVSHPEFSQSFGALDDTYYQTALAQINGQFPDATLLVFSDDPEWVRQHMFRER PHVLVENTGPDADVDDLQLMSLCHHHIIANSSFSWWGAWLNPRPDKRVIAPKQWFRNKPWNTADLIPAG WVRL Bacter- WP_ 495893515 glycosyl- 24.15 MRLIKMTGGLGNQMFIYAMYLKMKTIFPDVRIDLSDMVHYQVHYGYEMNKVFHLPRTEFCINRSLKKIIEFL 196 oidetes; 008618094.1 transferase LFKTILERKQGGSLVPYTRKYHWPWIYFKGFYQSEKYFAGIEKEVREAFVFDIRRASRRSLRAMQEIKADPHA Capno  [Bacter- VSIHVRRGDYLLEKHWKALGCICQSSYYLNALAELEKRVKHPHYYVFSEDLNWVRQNLPLIKAEFIDWNKGE cytophaga sp. oidetes] DSWQDMMLMSHCRHHIICNSTFSWWGAWLNGPLDKIVIAPERWTQTTDSADVVPESWLKVSIG oral taxon 329 str. F0087; Para- prevotella clara YIT 11840 Smarag- WP_ 516906936 protein 24.08 MADVVVTLAGGLGNQLFQTAYAKNLEARGHRVTLDGTVVRWTRGLHIDPQICGLKILNATPPAPVPGRLAA 197 dicoccus 018159152.1 [Smarag- TVLRRALATRLRFGPDGRIVTQRTLEFDEQYLNLNSPGRYRVEGYWQCERYFSDVGQTVRKVFLDMLGRH niigatensis dicoccus VSYNGLSRLPAMADPSSISLHVRRGDYVTANFIDPLALEYYERALEELAVPSPRIFVFSDDLDWATRELGRICD niigatensis] VIPVEPDWTSHPGGEIFLMSQCSHHIIANSSFSWWGAWLDGRTSSRVVAPRQWFLETYSARDIVPDRWT KV Bacteroides WP_ 547279005 family 11 24.05 MIHLILGGGLGNQMFQYAFARLSALQYNENISFNTILYKELKNEERSFSLGHLNINTMCIVETPDENKRIWELF 198 fragilis 022012576.1 glycosyl- NKQIFHQKIARKILPASIRWWWMSNRNIYANVCGPYKYYHPRHRSQNTTIIHGGFQSWKYFKEHQSMIKAE CAG:558 transferase LKVITPISEPNKKILKEIQNSNSICVHIRRGDFLSAQFSPHLEVCNKDYYEKAIKMISSQIENPTFFIFSNTHEDLV [Bacteroides WIRKNYNIPQNSVYVDLNNPDYEELRLMYNCKHFILSNSSFSWWAQYLSESKNKIIIAPKWDKRKGIDFSDIY fragilis  MPEWIIIK CAG:558] Desulfo- WP_ 550904402 protein 24.05 MSFSIDVAAIQRMALVKVDGGLGSQMWQYALSLAVGKKSSSFTVKHDLSWFRHYAKDIRGIENRFFILNSVFT 199 vibrio 022657592.1 [Desulfo- NINLRLASENERLFFHIALNRYPDSICNFDPDILALKQPTYLGGYYVNAQYVTSAEKEIREAYVFAPAVEESNQA vibrio] MLQTIHAAPMPVAVHVRRGDYIGSMHELVTPRYFERAFKILAAALQPKPTFFVFSNGMEWTKKAFAGLPYD FVYVDANDNDNVAGDLFLMTQCKHFTISNSLSWWGAWLSQRAENKTVIMPSKWRGGKSPIPGECMRV EHWHMCPVE Hoeflea WP_ 494373839 alpha-1,2- 24.05 MHGGLGNQLFQYAVGRAVALRTGSELLLDTREFTSSNPFQYDLGHFSIQAKVANSSELPPGKNRPLAYAW 200 photo- 007199917.1 fucosyl- WRKFGRSPRFVREQDLGYNARIETIEADCYLHGYFQSQKYFEDIASILWKDLSFRQAISGENASMAERIQSAP trophica; transferase SVSMHIRRGDYLTSAKARSTHGAPDLGYYGRALGEIRARSGSDPVVYLFSDDPDWVRNNMRMDANLVTVA Hoeflea [Hoeflea INDGKTAFEDLRLMSLCDHNIIVNSTFSWWGAWLNPSLDKIVVAPKRWFADPKLSNPDITPPGWLRLGD photo- photo- trophica DFL- trophica] 43 Vibrio WP_ 487957217 glycosyl 24.04 MKIISFSGGLGNQLFQYAFYLKDNSDFGNIFLDFSFYESQNKRDAVIRNFYGVDSLDIIKQSSYVRGKFLILKL 201 cholerae; 002030616.1 transferase, INKFRFFNNLLEFVDKENGLDETLLSTNKVFFDGYWQSYRYVKDYKSNIKELFSFYDFKGNILEVRKKICQSNSV Vibrio family 11 CMHVRRGDYVAEKNTKLVHGVCSLQYYRDALNNIKNVDNSIDHIFIFSDDIDWVKNNISFDIPVTVVDFVGQ cholerae O1 [Vibrio SVPDYAEMLLFSCGKHKVIANSTFSWWGAFLSDRNGVIVSPKKWFAKEEKNYDEIFIEGSLRL str. 87395 cholerae] Lachno- WP_ 551041074 protein 24.03 MIIVRFRGGMGNQMFQYAFLRYLEMKGATLKADLSEFKCMKTHAGYELDKAFDLHPAEASYKEIRAVADYI 202 spiraceae 022784718.1 [Lachno- PVMHRFPFSRKVFEILYKKEKRVEAEGPKKSHISEEKYFDMSEDERLHLASSSEDLYMDGFWIKPDMYDDE bacterium spiraceae VLKCFTFSKTLDEKYKGTIEDIHSCSVHVRCGDYTGTGLDILGKEYEKAAEKILSEDADVKFYVFSDDREKAEK NK4A179 bacterium LLSPFMKKMVFCDTPASHAYDDMYLMSRCRHHIIANSTFSFWGARLSADKSGITICPKYEDKNNTANRLVHE NK4A179 GWQML Cecembia WP_ 496476931 Glycosyl- 24.01 MIIMKFMGGLGNQIYQYALGRKLSELHNSFLASHIHIYKNDPDREFVLDKRNIKNKHLPWKVIKLLNSDYALKF 203 lonarensis; 009185692.1 transferase DKVFHTEFYHELVLEKALESKDIPRKNNLYLRGSWGNRKYYEDYIKDISDEITLKEKFKTKDFNTVNKKVKNSDS Cecembia family 11 VGIHIRRGDYEKVAHFKNFYGLLPPSYYSAAVDFIGNRIEKSNFFIFSDDTDWVKENLPFLKDSFFVSDIIGSVD lonarensis  [Cecembia YLEFELLKNCKHQIIANSTFSWWAARLNSNPAKIVIPKRWFADDRQQAVYEIEDSYYIKEAIKL LW9 lonarensis] Bacteroides WP_ 490423336 protein 24 MKIVNILGGLGNQMFVYAMYLALKEAHPEEEILLCRRSYKGYPLHNGYELERIFGVEAPEAALSQLARVAYPF 204 ovatus; 004295547.1 [Bacteroides FNYKSWQLMRHFLPLRKSMASGTTQIPFDYSEVTRNDNVYYDGYWQNEKNFLSIRDKVIKAFTFPEFRDEK Bacteroides ovatus] NKALSDKLKSVKTASCHIRRGDYLKDPIYGVCNSDYYTRAITELNQSVNPDMYCIFSDDIGWCKENFKFLIGDK ovatus ATCC EVVFVDWNKGQESFYDMQLMSLCHYNIIANSSFSWWGAWLNNNDDKVVVAPERWMNKTLENDPICDN 8483 WKRIKVE Bacteroides WP_ 547668508 glycosyl- 23.99 MRLIKMTGGLGNQMFIYAFYLKMKKLFPHTKIDLSDMMHYHVHHGYEMNRVFALPHTEFCIBNRTLKKLME 205 coprocola 022125287.1 transferase FLLCKVVYERKQKNGSMEAFEKKYAWPLIYFKGFYQSERFFADIEDDVRKTFCFNMELINSRSREMMKIIDAD CAG:162 family 11 EHAVSIHIRRGDYLLPKFWANAGCVCQLPYYKNAITELEKHESTPSFYVFSDDIEWVKQNLSLPNAHYIDWN [Bacteroides QGNDSWQDMMLMSHCRNHIICNSTFSWWGAWLNPRKNKTVIVPSRWFMKEETPIYPVSWIKVPIN coprocola CAG:162] Bacteroides WP_ 495110765 gylcosyl- 23.99 MRLIKVTGGLGNQMFIYAFYLRMKKYYPKVRIDLSDMMHYKVHYGYEMHRVFKLPHTEFCINQPLKKIIEFLF 206 dorei; 007835585.1 transferase FKKIYERKQAPNSLRAFEKKYFWPLLYFKGFYQSERFFADIKDEVREAFTFDRSKANSRSLDMLDILDKDENAV Bacteroides [Bacteroides SLHIRRGDYLQPKHWATTGSVCQLPYYQNAIAEMSKRVTSPSYYIFSDDIVWVRENLPLQNAVYIDWNTGE dorei DSM dorei] DSWQDMMLMSHCKHHIICNSTFSWWGAWLNPSIDKTVIVPSRWFQYSETPDIYPTGWIKPVD 17855; Bacteroides dorei CL02T12C01 Bacteroides; WP_ 494936920 protein 23.97 MIIVRLWGGLGNQLFQYSFGQYLEIETDKKVFYDVASFTSDQLRKLELCSFIPDIPLYNAYFTRYTGVKNRLF 207 Bacteroides 007662951.1 [Bacteroides] KALFQWSNTYLSESMFDICLLEKARGKIFLQGYWQEEKYATYFPMQKVLSEWKNPNVLSEIEENIRSAKISVS intestinalis LHVRRGDYFSPKNINVYGVCTEKYYEQAIDRANSEIEEDGQFFVFSDDILWVKNHVSLPESTVFVPNHEISQFA DSM 17393; YIYLMSLCKVNIISNSTFSWWGAYLNQHKNQLVIAPSRWTFTSNKTLALDSWTKI Bacteroides intestinalis CAG:564 Lachno- WP_ 511028838 protein 23.95 MIVIHVMGGLGNQLYQYALYEKLRALGREVKLDVYAYRQAEGAEREWRALELEWLEGIRYEVCTAAERQQL 208 spiraceae 016283022.1 [Lachno- LDNSMRLADRVRRRLTGRRDKTVRECAAYMPEIFEMDDVYLYGFFWGCEKYYEDIIPLLQEKIVFPESSNPKN bacterium A4 spiraceae ADVLRAMAGENAVSVHIRRKDYLTVADGKRYMGICTDAYYKGARFYITERVERPVFYIFSDDPAFAKTQFCE bacterium A4] ENMHVVDWNTGRESLQDMALMSRCRHNICANSTFSIWGARLNRHPDKIMIRPLHHDNYEALDARTVHEY WKGWVLIDADGKV Phaeobacter YP_ 399994425 protein 23.91 MIITRLHGRLGNQMFQYAAGRALADRLGVSVALDSRGAELRGEGVLTRVFDLDLATPDILPPLRQRAPLGYA 209 gallaeciensis; 006574665.1 PGA1_ LWRGLGQHLGTGPKLRREVGLGYNPDFVDWSDNSYLHGYWQSERYFAWSAERIRRDFTFPEYSNQQNAE Phaeobacter c33070 MAARIGETNAISLHVRRDGYLTLAAHVKCDQAYYEAALAQVLDGLEGQPTVYVFSDDPQWAKENLPLCDK gallaeciensis [Phaeobacter VVVDFNGADTDYEDMRLMSLCKHNIIGNSSFSWWAAWLNGTPDRRVAGPTKWFGDPKLNNPDILPPDW DSM 17395 = gallaeciensis LRISV CIP 105210 DSM 17395 = CIP 105210] Firmicutes WP_ 546362318 protein 23.88 MSGGLGNQMFQYALYLKLRSLGREVCFDDKSQYDEETFRNSSQKRRPKHLDIFGITYPSAGKEELEKLTDGA 210 bacterium 021849028.1 [Firmicutes MDLPSRIRRKILGRKSLEKNDRDFMFDPSFLEETEGYFCGGFQSPRYFAGAEEEVRKAFTFPEELLCPKEGCSR CAG:791 bacterium EQEKMLEQSASYAERIRKANCEAADRGVPGGGSASIHLRFGDYVDKGDIYGGICTDAYYDTAIRCLKERDPG CAG:791] MIFFVFSNDEEKAGEWIRYQAERSENLGRGHFVLVKGCDEDHGYLDLYLMTLCRNHVIANSSFSWWASFM CDAPDKMVFAPSIWNNQKDGSELARTDIYADFMQRISPRGTRLSDRPLISVIVTAYNVAPYIGRALDSVCGQ TWKNLEIIAVDDGSSDETGAILDRYAAGDSRIQVVHTENRGVSAARNEGIAHARGEYIGFVDGDDRAHPAM YEAMIRGILSSGADMAVVRYREVSAEETLTDAEEQVASFDPVLRASVLLQQRDAVQCFIRAGMAEEEGKIVL RSAVWNKLFHRRLLRDNRFPEGTSAEDIPFTTRALCLSKKVLCVPEILYDYVVNRQESIMNTGRAERTLTQEIP AWRTHLELLKESGLSDLAEESEYWFYRRMSLYEEEYRRCSETAKEAKELQERILKHRDRILELAEEHSFGRRGD RERLKLYVNSPRQYFLLSDLYEKTVVNWKNRPDKT Butyrivibrio WP_ 302669773 glycosyl- 23.84 MRKRIIALNGGLGNQMFQYAFARMLEDRKHCLIEFDTGFYSTVNDRKLAIQNYNIHKYDFCNHEYYNKIRLLF 211 proteo- 003829733.1 transferase QKIPFVAWLAGTYKEYSEYQLDPRVFLFNYRFYYGYWQNKQYFENISNDIRNELSYIGNVSEKENALLNMLEA clasticus; 11 HNAIAIHVRRGDYTQEGYNKIYISLSEKEYYKRAVSIACKELGDNNIPLYVFSDDIDWCKANLADIGNVTFVDNT Butyrivibrio [Butyrivibrio ISSSADIDMLMMKKSRCLITANSTFSWWSAWLSDRDDKIVLVPDKWLQDEEKNTKLMKAFICDKWKIVPV proteo- proteo- clasticus clasticus B316 B316] Bacteroides WP_ 496043738 protein 23.81 >gi|496043738|ref|WP_008768245.1|protein[Bacteroides sp. 212 sp. 008768245.1 [Bacteroides 2_1_16]MQVVARIIGGLGNQMFIYATARALALRIDADLILDTQSGYKNDLFKRNFLLDSFCISYRKANCFQKY 2_1_16 sp. DYYLGEKVKSLGKKTHFSVIPFMKYISENTSCDFVDGLLKKHILSVYLDGYWQNEAYFKDYASIIKKDFQFCQV 2_1_16] NDLRTLSEAEIIKKSITPVAIGNRRYQELNSHQNTKVTDLDFYQKAINYIESKVDMPTFFIFSEDQEWVKNNLEQ KSNFIMISPKEGNYSALNDMYLISLCKHHIVSNSSFYWWGAWLANNKNKIVVASDCFLNPQSIPDSWIKF Desulfo- YP_ 256830317 glycosyl- 23.76 >gi|256830317|ref|YP_003159045.1|gylcosyl transferase family protein 213 microbium 003159045.1 transferase [Desulfomicrobium baculatum DSM] baculatum; family protein MAKIVTRIMGGIGNQLFCYAAARLALVNHAELVIDDVTGFSRDRVYRRRYMLDHFNISARKATNYE Desulfo- [Desulfo- RMEPFERYRRGLAKYISKKLPFFEREYIEQERIEFDPRFLEYRTYNNIYIDGLWQSENYFKDVEDIIRDDLKIIPPT microbium microbium DLENINIAKKIKNIQNTIAMHVRWFDLPGINLGNNVSTYYYHRAIAMMEQRINAPHYFLFSDNLEAVHSKLD baculatum baculatum LPEGRVTFVSNNDGDDNAYADLWLMSQCKHFITANSTFSWWGAWLGESRDSVVLVPRFSPDGGVTSWC DSM 4028 DSM 4028] FTGLIPERWEQVSSIR Prevotella WP_ 545304945 galactoside 23.76 MDIVLIFNGLGNQMSQYAFYLAKRQRNNHTVYCHFGPRTQYSLDKLFDIPYRHNAVLVLLYRALDHAHFSN 214 pleuritidis; 021584236.1 2-alpha-L- HRWLRRLLRPTLQLLGVKMIVERPSRDFDMRHFTHQKGIVFYRGGWHSELNFTAVADAVKRRFRFPEIQDA Prevotella fucosyl- AVLAVIDRIKSCQSVSLHLRRGDYLSLSEFQFVCTEAYYEHAIAYFESQIESPEYFVFSDDPTYAREQFGADPNF pleuritidis transferase HIIDLNHGEDAWCDLLMMTQCRYNIIASNTFSWWGAWLNDNPSKIVVHPRYHLNGVETRDFYPRNWICIE F0068 [Prevotella pleuritidis] Bacteroides WP_ 496038684 glycosyl- 23.75 MKVIWFNGNLGNQVFYCKYKEFLHNKYPNETIKYYSNSRSPKICVEQYFRLSPDRIDSFKVRFVFEFLGKFFR 215 sp. 1_1_14 008763191.1 transferase, RIPLKVPKWYCTRKSLNYEASYFEHYLQDKSFFEKEDSSWLKAKKPDNFSEKYLIFENLICNTNSVANHIRRGD family 11 YIKPGSDYEDLSATDYYEQAIKKATEVYLDSQFFFFSDDLEFVKNNFKGDNIYYVDCNRGADYLDILLMSQAK [Bacteroides INIIANSTFSYWGAYMNHEKKKVMYSDLWFRNESGRQPNIMLDSWICIETKRK sp. 1_1_14] Agromyces WP_ 551273588 protein 23.65 MVGRVGIARRQAADVSCTDGDGLVAWRIRTGEIVLGLQGGIGNQLFEWAFAMALRSIGRRVLFDAVRCRG 216 subbeticus 022893737.1 [Agromyces DRPLMIGPLLPASDWLAAPVGLALAGATKAGLLSDRSWPRLVRQRRSGYDPSVLERLGGTSYLLGTFQSARY subbeticus] FDGVEHEVRAAVRALLEGMLTPSGRRFADELRADPHRVAVHVRRGDYVSDPNAAVRHGVLGAGYYDQAL EHAAALGHVRRVWFSDDLDWVREHLARDDDLLCPADATRHDGGEIALIASCATRIIANSSFSWWGGWLG APSSPAHPVIAPSTWFADGHSDAAELVPRDWVRL Prevotella WP_ 494220705 alpha-1,2- 23.59 MIATTLFGGLGNQMFIYATAKALSLHYRTPMAFNLRQGFEQDYKYQRHLELNHFKCQLPTAKWITFNYKGE 217 salivae; 007133870.1 fucosyl- LNIKRISRRIGRNLLCPHYQFIKEKEPFHYEKRLFEFTNKKIFLEGYWQSPRYFENYSDEIRRDFQLKSILPHTITD Prevotella transferase ELQMLKGTGKPLVMLGIRRYQEVKDKKDSPYPLCNKDYYAKAISHVQEQLPAPLFVVFTQEQAWAMNNLP salivae [Prevotella TNANLYFVKEKDNAWATIADMYLMTQCQHAIISNSTFYWWGAWLQHPIENHIVVAPNNFINRDCVCDN DSM 15606 salivae] WIILD Carno- YP_ 554649641 glycosyl- 23.57 MIFVDLSEGLGNQMFQYAYSRYLQELYGGTLYNTSSFKRKNSTRSYSLNNFYLYENVKLPSKFRRVIYNFYSK 218 bacterium sp. 008718687.1 transferase TIRMFIKKVIRMNPYSDKYYFSMIPYGFYVSSQVFKYLTVPTTKRHNIFMGTWQTNKYFQSINDKIKDELKV WN1359 [Carno- KTEPNELNKKLITEINSNQSVCVHIRLGDYTNPEFDYLVCTSDYYLKGMDYIVSKVKEPNFYIFSNSSSDIEWI bacterium sp. KNNFYFKYKVKYIDLNNPDFEDFRLMYNCHHFIISNSTFSWWAQFLSNNDKKIIVAPSKWQKSNENEAKDIY WN1359] LDHWKLIEIE Butyrivibrio WP_ 551018062 glycosyl- 23.55 MLIIQIGGLGNQMQQYAYLEKFKALGKETRLDTSWFDNASMQENVLARRSLELRFFDNLTYEACTPQERA 219 sp. AD3002 022762290.1 transferase RFTDSVARVVEKLVPGMGSRFTESCMYHPEIFELKDKYIEGYFACQKYYDDIMGELQELFVFPTHPDEEINI [Butyrivibrio KNMNLMNEMEMVPSVSVHIRRGDYLDPENAALFGNIATDAYYDSAMEYFKAIDPDTHFYIFTNDPEYAREK sp. AD3002] YADPGRYTIVDHNTGKYSLLDIQLMSHCRGNICANSTFSFWGARLNRRKDKIPVRTLVMRNNQPVTPELMH EYWPGWVLVDKDGKVR Clostidium WP_ 454396682 gycosyl- 23.55 MIVIRVMGGLGNQMQQYALYEKFKALGKETRLDTSWFDNASMQENVLARRSLELRFFDNLTYEACTPQER 220 sp. KLE 1755 021636935.1 transferase, EALLGKEGFFNKLERKLFPSKNKHFYESEMFHPEIFKLDNVYLEGHWACEKYYHDIMPLLQSKIIFPKTDNIQN family 11 NMLKNKMNSENSVSIHIRRGDYLDPENAAMFGGICTDSYYKSAEGYIRNRVTNPHFYLFSDDPAYLREHYKG [Clostidium EEYTVVDWNHGADSFYDMELEMSCCKHNVCANSTFSFWGARLRTEKKIVIRPAKHKNSQQAEPERMHEL sp. KLE 1755] WENWVIIDEEGRIV Bacteroides; YP_ 150005950 glycosyl- 23.47 MKFFVFGGGLGNQLFQYSYYRYLKKKYPSERILGIYPDSLKAHNGIEIDKWFDIELPPTSYLYNKLGILLYRVNRF 221 Bacteroides 001300694.1 transferase LYNGHYRLLFCNRVYPQSMKHFFQWGDWQDYSIIKQINIFEFRSELPIGKENMEFLKKMETCNSISVHIRRG vulgatus ATCC  family protein DYLKTDLIHIYGGICTSKYYREAIKFMEQEVEEPFFFFFSDDCLYVETEFADIRNKIIISHNRDDRSFFDMYLMAH 8482; [Bacteroides AKNMILANSTFSCWAAYLNRTAKIIITPDRWVNTDFSKLEALPNEWIKIRV Bacteroides vulgatus dorei DSM ATCC 8482] 17855; Bacteroides massiliensis dnLKV3 Para- WP_ 495902050 glycosyl- 23.47 MRLIKMTGGLGNQMFIYAMYLKMRAVFPDTRIDLSDMVHYRVHYGYEMNKVFNLPRTEFRINRSLKKIIEFL 222 prevotella 008626629.1 transferase LFKTILERKQGGSLVPYIRKYHWPWIYFKGFYQSEEYFAGVEKEVREAFVFDVRRVNRKSLCAMQEIMADPD xylaniphila; [Para- AVSIHVRRGDYLQGKHWKSLGCICQRSYYLNALSELEKRIVHPHYYVFSEDLDWVRQYLPLENAVFIDWNKG Para- prevotella EDSWQDMMLMSHCRHHIICNSTFSWWGAWLNPSPDKIVIAPERWTQQTTNSADVVPESWLKVSIG prevotella xylaniphila] xylaniphila YIT 11841 Thauera WP_ 489020296 glycosyl- 23.47 MTDRALIAIVKGGLGNQLFIYAAARAMALRTGRQLYLDAVRGYLADDYGRSFRLNRFPIEAELMPEQWRVA 223 sp. 28 002930798.1 tansferase STLRHPRAKLVRALNKYLPEAWRFYVAEFGTRPGALWNHGRNVKRVTLMGYWQDEAYFLDYAELLRREL family GPPMPDAPEVRARGEFFAGTESVFLHVRRCRYSPLLDAGYYQKAVDLACAELNKPVFMIFGDDIEWVVNNI protein DFRGAGYERQDYDESDELADLWLMTRCRHAIIANSSFSWWAAWLGGAAGSGRHVWAPGQSGLALKCAK [Thauera SWEAVDAQPE sp. 28] Subdo- WP_ 494107522 alpha-1,2- 23.44 MIYAELAGGLGNQMFIYAFARALGLRCGEAVTLLDRQDWRDGAPAHTACALEGLNLVPEVKILAEPGFAKR 224 ligranulum 007048308.1 fucosyl- HLPRQNTAKALMIKYEQRQGLMARDWHDWERRCAPVLNLLGLHFATDGYTPVRRGPARDFLAWGYFQS variabile; transferase EAYFADFAPTIRAELRAKQAPAGVWAEKIRAAACPVALHLRRGDYCRPENEILQVCSPAYYARAAAAAAAAY Subdo- [Subdo- PEATLFVFSDDIDWAKEHLDTAGLPAVWMPRGDAVGDLNLMALCRGFILSNSTYSWWAQYLAGEGRTV ligranulum ligranulum WAPDRWFAHTKQTALYQPGWHLIETR variabile variabile] DSM 15176 Firicutes WP_ 547127527 protein 23.4 MIIVEVMGGLGNQMQQYALYRKLESLGKDARLDVSWFLDKERQTKVLASRKLELSWFENLPAKYCTQEEK 225 bacterium 021916223.1 [Firmicutes QAILGKNNLIGKLKKKLLGGSNRHFTESDMYHPEIFDLEDAYLSGFWACEAYYADILPMLRSQIHFPDPEKGE CAG:24 bacterium GWDLEAAAKNKETMERMKQETSVSIHIRRGDYLDAKNAEMFGGICTDAYYEAAISYIKEQTPDAHFYVFSD CAG:24] DSAYVKNAYPGKEFTVVDWNTGKNSLFDMQLMSCCNHNICANSTFSWGARLNPSPDKVMIRPSKHKNS QNIVPEEMKRLWDGWVLIDGKGRII Prevotella WP_ 547906803 glycosyl- 23.39 MIITKLNGGLGNQLFEYACARNLQLKYNDVLYLDIEGFKRSPRHYSLEKFKLSSDVRMLPEKDSKSLILLQAISK 226 sp. CAG:474 022310139.1 transferase LNRNLAFKLGPLFGTYIWKSSNYRPLKIKNTRGKKLYLYGYWQSYEYFKENEAIIKQELNVKETIPIECSELLKEIN family 11 KPHSICVHVRRGDYVSCGFLHCDEAYYNRGINHIFDKHPDSNVVVFSDDIKWVKANMNFDHPVAYVEVDV [Prevotella PDYETLRLMYMCKHFVMSNSSFSWWASYLSDNKEKIVVAPSYWLPANKDNKSMYLDNWTIL sp. CAG:474] Roseburia WP_ 493910390 gylcosyl- 23.38 intestinalis]MRGNRGMIAVKIGDGMGNQLFNYACGYAQARRDGDSLVLDISECDNSTLRDFELDKFHLKY 227 intestinalis; 006855899.1 transferase DKKESFPNRNLGQKIYKNLRRALKYHVIKEREVYHNRDHRYDVNDIDPRVYKKKGLRNKYLYGYWQHLAYFE Roseburia family 11 DYLDEITAMMTPAYEQSETVKKLQEEFKKTPTCAVHVRGGDIMGPAGAYFKHAMERMEQEKPGVRYIVFT intestinalis; [Roseburia NDMERAEEALAPVLESQKKDAVGQAENRLEFVSEMGEFSDVDEFFLMAACQNQILSNSTFSTWAAYLNQN L1-82 intestinalis] PDKTVIMPDDLLSERMRQKNWIILK Bacteroides WP_ 490424433 protein 23.29 MKIVLFTPGLGNQMFQYLFYLYRDNYPNQNIYGYYNRNILNKHNGLEVDKVFDIQLPPHTVISDASAFFIRA 228 ovatus; 004296622.1 [Bacteroides LGGLGLKYFIGKDQLSPWKVYFDGYWQNKEYFQNNVDKMRFREGFLNKKNDDILSLIRNTNSVSNHVRRG Bacteroides ovatus] DYCDSCRKDLFLQACTPQYYESAISVMKEKFQKPVFFVFSDDIPWVKVNLNIPNAYYIDWNKKENSYLDMYL ovatus ATCC MSLCTASIIANSTFSFWGAMLGNKKELVIKPKKWIGDEIPEIFPPSWLSL 8483 Butyrivibrio WP_ 551035785 gylcosyl- 23.25 MLIIQIAGGLGNQMQQYALYRKLLKYHPDGVRLDLSWFDSEVQKNMLAKREFELALFKGLPYIECKPEERAA 229 sp. AE3009 022779599.1 transferase FLDRNAAQKLSGKVLKKLGLRDNANPNVFEESRMFHPEIFELDNKYIIGYFACQKYYDDIMGDLCNLFEFPEH [Butyrivibrio LDPELEKKNLELISKMEKENSVSVHIRRGDYLDPENFKILGNIATDEYYESAMKYFEDRYEKVHFYIFTSDHEYA sp. AE3009] REHFADESKYTIVDWNTGKDSLQDVRLMNHCKGNICANSTFSWGARLNQRQDKVMIRTYKMRNNQPV DPDTMHDYWKGWILIDETGREV Butyrivibrio WP_ 302669752 glycosyl- 23.23 MTKNEKKLIVKFQGGLGNQLYEYAFCEWLRQQYSDYEVLADLSYYKIRSAHGELGIWNIFPNINIEVASNWDI 230 proteo- 003829712.1 transferase 11 IKYSDQIPIMYGGKGADRLNSVRTNVDRFFSKRKHSYYTEISNTDVSEVINALNNGIRYFDGYWQNIDYFKG clasticus; [Butyrivibrio NIEDLRNKLKFSEKCDKYITDEMLRDNAVSLHVRRGDYVGSEYEKEVGLSYYKKAVEYVLDRVDQAKFFIFSD Butyrivibrio proteo- DKYYAETAFEWIDNKTVVAGYDNELAHVDMLLMSRMKNNIIANSTFSLWAAYLNDSMNPLIVYPDVESLD proteo- clasticus KKTFSDWNGIIK clasticus B316] B316 Prevotella WP_ 517173838 protein 23.23 MKSQFLKHIKLSGGFGNQLFQYFFGEYLKEKYNCSISFFSEPALDINQLQIHRFFPALRISHNTELRPYHYSFTQ 231 nanceiensis 018362656.1 [Prevotella QLAYRCMRKLLLLFPFLNRVKIENGSNYQNQSFNDTYCFFDGYWQSYRYLSAFTPSLQFEDQLINDISADYIN nanceiensis] AIEQSEAVFLHIRRGDYLNKENQKVFACECPLNYFENAANRIKEDIKNVHFFVFSNDIQWVKSHLKLNDNEVTF IQNEGNSCDLKDFYLMTRCKHAIISNSTFSWWAAYLINNSDKKVIAPKHWYNDISMNNAATKDLIPPTWIRL Ruegeria sp. WP_ 495838392 aplha-1,2- 23.23 MIITRLHGRLGNQMFQYAAGRALADRAGVPLALDSRGAILRGEGVLTRVFDLELADPVHLPPLKQTNPLRYA 232 R11 008562971.1 fucosyl- IWRGIGQKVGAKPYFRRERGLGYNPAFEDWGDSNYLHGYWQSQKYFQNSAERIRSDFTFPAFSNQQNAE transferase MAARIAESTAISLVHVRRGDYLTFAAHVLCDQAYYDAALAKVLDGLQGDPIVYVFSDDPQWAKDNLSLPCEK [Ruegeria sp. VVVDFNGPETDFEDMRLMSLCQHNIIGNSSFSWWAAWLNQTPGRRVAGPAKWFGDPKLSNPDIFPHDW R11] LRISV Winograd- WP_ 527072096 aplha-1,2- 23.21 MGNQLYEYATAKAMAVALNKKLVIDPRPILKEAPQRHYDLGLFNIQDEDFGSPFVQWLVRWVASVRLGKFF 233 skyella 020895733.1 fucosyl- KTIMPFAWSYQMIRDKEEGFDESLLQQKSRNIVIEGYWQSFKYFESIRPTLLKELSFKDKPNAINQKYLDEISV psychro- transferase NAVANHIRRGDYVANPVANAVHGLCDMDYYKKAIAIIKDKVENPYFFIFTDDPDWAEKNFKISEHQKIIKHNI tolerans RS- [Winograd- GKQDHEDFRLLTNCKYFIIANSSFSWWGAWLSDYKNKIVISPNKWFNVDAVPITERIPESWIRV 3; Winograd- skyella skyella psychro- psychro- tolerans] tolerans  Lachno- WP_ 551041720 protein 23.2 MITVRIDGGFGNQMFQYAFFLHLKKTITDNKISVDLNCYNPHGSGDIFTRFKLAPEQAAPSEIKRFHRNSIYHL 234 spiraceae 022785341.1 [Lachno- LRPLDSAGITTNPYYREEDIDDLNSVLNKKRVYLRGYWQDKRYPFSVKDQLIDCFDLGKMDMTGASAENNVI bacterium spiraceae LEQIASEESRSVGBHLRGGDYIGDPVYSGICTPEYYEAAFKHVSEKIKDPVFHIFTNDISMIEKCGLSGKYDLKIT NK4A179 bacterium DINDEAHGWADLKLMSACRHHIISNSSFSWWAAFLGEATTEASADVINVIPEYMRQGVSAETLRCPCWTT NK4A179] VTSDGRVYPS Prevotella WP_ 496522549 aplha-1,2- 23.13 MKIVCIKGGLGNQLFEYCRYRSLHRHDNRGVYLHYDRRRTKQHGGVWLDKAFHITLPNEPLRVKLLVMVLK 235 sp. oral taxon 009230832.1 fucosyl- TLRRLHLFKRLYREEDPRAVLIDDYSQHKQYITNAAEILNFRPFEQLDYAEEIQTTPFAVSVHVRRGDYLLLANK 317 str. transferase SNFGVCSVHYYLSAAVAVRERHPESRFFVFSDDMEWAKENLNLPNCVFVEHAQAQPDHADLYLMSLCKGH F0108; [Prevotella IIANSTFSFWGAYLSKGSSAIAIYPKQWFAEFTWNVPDIFPAHWMAL Prevotella sp. oral taxon sp. oral taxon 317] 317 Butyrivibrio WP_ 551021633 glycosyl- 23.1 MLIIQIAGGLGNQMQQYAVYTKLRGMGKDVRLDLSWFDPSVQKNMLAPREFELSMFEGVDYTECTAEER 236 sp. XPD2006 022765796.1 transferase DSFLKQGMIANVTGKMLKKLGLRDEANPKVFSEKEMYHPEIFELEDRYIKGYFACQKYYDDIMGELWEKYTF [Butyrivibrio PAHSDPDLHTRNMALVERMEKETSVSVHIRRGDYLDPSNVEILGNIATEEYYQGAMDYFSVKDPDTHFYIFT sp. XPD2006] SDHEYAREKFSDESKYTIVDWNSGRNSVQDLMLMSHCKGNICANSTFSFWGARLNRRPDKTVIRTYKMRN NQPVNPDIMHDYWKGWILMDEGSII Butyrivibrio WP_ 551008140 protein 23.08 MIIIKLQGGLGNQLFLYGLYKNLKHLKRDVKMDIESGFEEDKLRVPCLKSMGLDYEVATRDEIVAIRDSYMDIF 237 fibrisolvens 022752717.1 [Butyrivibrio SRIRRKITGRKTFDYYEPEDGNFDPRVLEQTHAYLDGYFQSEKYFGDSDDRKKLKDELLKEKIRVLDSSDTLKDL fibrisolvens] YNMMSSGSSVSLHIRRGDYLTPGIMETYGGICTDEYYDIAMNRIKNEYPDSKFFIFSNDIDWCKEKYGSRDDV IFVDSCDEHEGLTNVSGDQDDIQVQGDIKEHGNNSLRDAAELYLMSACKHHILANSSFSWWGAWLSDHEG MTIAPSKWLNNKNMTDIYTKDMLLI Cylindros- WP_ 493321658 Glycosyl- 23.05 MKKTVVLLKGGLGNQMFQYAFARSISLKNSSKLVIDNWSGFTFDYKYHRQYELGTFSIVGRPANLTEKFPFW 238 permopsis 006278973.1 transferase FYELKSKFFPRLPKVFQQQFYGLLINEVGGEYIPEIEETKISQNCWLNGYWQSPLYFQKHSDSITRELMPPEPM raciborskii; family 11 EKHFLELGKLLRETESVALGIRLYEESKNPGSHSSSGELKSHFEINQAILKLRELCNGAKFFVFCTHRSPLLQELAL Cylindros- [Cylindros- PENTIFVTHDDGYVGSMERMWLLTQCKHHIFTNSTFYWWGAWLSQKFYIQGSQIVFAADNFINSDAIPKH permopsis permopsis WKPF raciborskii raciborskii] C5-505 Prevotella WP_ 494609908 alpha-1,2- 23.05 MKIVNFQGGLGNQMFIYAFSRYLSRLYPQEKIYGSYWSRSLYVHSAFQLDRIFSLQLPPHNLFTDCISKLARFF 239 multiformis; 007368154.1 fucosyl- ERLRLVPVEETPGSMFYNGYWLDKKYWEGIDLSEMFCFRNPDLSAEAGAVLSMIERSNAVNVHIRRGDYQS Prevotella transferase EEHIEKFGRFCPPDYYRIATERIRQREDDPLFFVFSDDMMWVKSNMDVPNAVYVDCHHGDDSWKDMFL multiformis [Prevotella MAKCRHNIIANSTFSFWAAMLNANPDKVVVYPQRWFCWPSPDIFPEMWLPVTEKEIKSSF DSM 16608 multiformis] Bacteroides WP_ 548151455 protein 23 MIIVNMACGLANRMFQYAFYLSLKERGYNVKVDFYKSATLPHENVPWNDIFPYAEIDQVSNFRVLILGGGA 240 sp. CAG:462 022384635.1 [Bacteroides NLLSKLRRKYLPSLTNVITMSTAFDTDLQIDDDRKDKYIIGVFQSAAMVEGVCKKVKQCFSFLPFTDLRHLQLE sp. CAG:462] KEMQECESVAIHVRKGNDYQQRIWYQNTCTMDYYRKAIAEIKGKVKDPRFYVFTDNADWVRRNFTDFDYK MVEGNPVYGWGSHFDMQLMSRCKYNIISNSTYSWWGAYLNANRNKIVICPNIWFNPESCNEYTSCKLLCK GWIAL Desulfo- WP_ 492830222 Glycosyl- 23 MRIGILYICTGKYTVFWNHFFTSCEQHFLREHEKHYYIFTDGEIAHLNCNRVHRIEQQHLGWPDSTLKRFHM 241 vibrio 005984176.1 transferase FERIADTLRQNSDFIVFFNANMVFLRDVGKEFLPTREQALVFHRHPGLFRRPAWLPYERRPESTAYIPYGSG africanus; family 11/ SIYVCGGVNGGYTQPYLDFVAMLRRNIDIDVERGIIARWHDESHINRFVIGRHYKIGHPGYVYPDRRNLPFPR Desulfo- Glycosyl- IIRVIDKASVGGHTFLRGQTPEPAPEEQSKTVAKKLRSQLKRPCMPRAAQDEPIILARMMGGLGNQMFIYAA vibrio transferase ARVLAERQGAQLHLDTGKLSGDSIRQYDLPAFSIDAPLWHIPCGCDRIVQAWFALRHVAAGCGMPKPTMQ africanus PCS family 6 VLRSGFHLDQRFFSIRHSAYLIGYWQSPHYWRGHEDRVRSSFDLTRFERPHLREALAAVSQPNTISVHLRRG [Desulfo- DFRAPKNSDKHLLIDGSYYERARKLLLEMTPQSHFYIFSDEPEEAQRLFAHWENTSFQPRRSQEEDLLLMSRC vibrio SASIIANSSFSWWGAWLGRPKQHVIAPRMWFTRDVLMHTYTLDLFPEKWILL africanus] Roseburia sp. WP_ 548374190 protein 22.98 MILIHVMGGLGNQLYQYALYEKMKSLGKKVKLDTYAYNDAAGEDKEWRSLELDRFPAIEYDKATSEDRTKLL 242 CAG:100 022518697.1 [Roseburia sp. DNSGLLTAKIRRKLLGRKDKTIRESKEYMPEIFHMDDVYLYGFWNCERYYEDIIPLLQDKLQFPISNNPRNQQ CAG:100] CIEQMQKENAVSIHIRRTDYLTVADGARYMGICTEDYYKGAMAYIEERVSNPVYYIFSDDVEYAKQHYHQD NMHVVDWNSKADSIYDMQLMSKCKHNICANSTFSMWAARLNQNEKIMIRPLHHDNYETTTATQVKQN WKNWILLDQNGQVCE Lachno- WP_ 550997676 protein 22.96 MTMNIIRMSGGLGSQMFQYALYLKLKSMGKEVFDDINEYRGEKARPIMLAVFGIEYPRATWDEITSFTDG 243 spiraceae 022742385.1 [Lachno- SMDLLKRLRRKIFGRKAIEYEEQGFYDPNVLNFDSMYLRGNFQSEKYFQDIKEEVRKLYRFSTLEDMRLPERLY bacterium spiraceae KATKACLDGIESSESVGLHMYRSDSRVDGELYDGICTGNYYKGAVRFIQDKVPDAKFYIFSNEPKWVRGWVV 10-1 bacterium DLIQSQIQEGMSPSQVKEMEKRFVMVEANTEYTGYLDMMLMSKCKHNIISNSSFSWWSAWMNDHPEKV 10-1] WWAPDRWSSDKEGNEIYTTGMTLVNEKGRVNYIHENSTVK Prevtotella WP_ 490496500 protein 22.96 MILSYITGRLGNQLFEYAYARSLLLKRGKNEELILNFSLVRAAGKEIEGFDDNLRYFNVYSYTELDKDIVLSKGDL 244 nigrescens; 004362670.1 [Prevotella LQLFIYILFKLDQKLFRIIKEKWFSFFRRFGIIFQDYLDNISNLIIPRTKNVFCYGKYENPKYFDDIRSILLKEFTP Prevtotella nigrescens] PPLKNNDQLYSVIESTNSVCISIRRDFLCDKFKDRFLVCDKEYFLEAMEEAKKRISNSTFIFFSDDIEWVRENIH nigrescens SDVPCYYESGKDPVWEKLRLMYSCKHFIISNSTFSWWAQLSRNEEKVVIAPDRWSNVPGEKSFLLSNSFIKI F0103 PIGILP Bacteroides WP_ 547952493 fucosyl- 22.95 MIYVEINGRLGNNMFEIAAAKSLTDEVTLWCKGDWQLNCIKMYSDTLFKNYPIVKSLPNNIRIYEEPEFTFHPI 245 sp. CAG:875 022353235.1 transferase PYKENQDLLIKGYFQSYKYLDREKVLKLYPCPMPVKLDIEKRFGDILSQYTVVSINVRRGDYLNLPHRHPFVGK [Bacteroides KFLERAMLWFGDKVHYIISSDDIEWCKAHFKQFDNVHYLTNSYPLLDLYIQTACHHNIISNSSFSWWGAYLN sp. CAG:875] NHPQKIVIAPHRWFGMSTNINTQDLLPPEWMIEQCVYEPKVFLKALPLHAKYLLKRVLK Prevotella YP_ 532354444 protein 22.9 MDSQLLKHIKLSGGFGNQLFQYFFGEYLKEKYNCSISFFSEPALDINQLQIHRFFPTLRISHNTELRRFHYAFTQ 246 sp. oral 008444280.1 HMPREF0669_ QLAYRCMRKLLLLFPFLNRKVKIENGSNYQNQSFNDTYCFDGYWQSYRYLSAFTPSLQFEDQLINDISADYIN taxon 299 str. 00176 AIEQSEAVFLHIRRGDYLNKENQKVFAECPLNYFENAVNKIKEGNKTYHFFVFSNDIEWVKCHLKLNNNEVTF F0039; [Prevotella IQNEGSSCDLKDFYLMTRCKHAIISNSTFSWWAAYLINNNDKKVIAPKRWYNDLSMNNATKDLIPPTWIRL Prevotella sp. oral sp. oral taxon 299 taxon 299 str. F0039] Para- WP_ 495904204 alpha-1,2- 22.87 MKIVCLKGGLGNQMFEYCRFRDLMDSGNGKVYLFYDRRRLKQHDGLRLSDCFELELPSCPWGIRLVVWGL 247 prevotella 008628783.1 fucosyl- KICRAIGVLKRLYDDEKPDAVLIDDYSQHRRFIPNARRYFSFRQFLAELQSGFVQMIRAVDYPVSVHVRRGDY xylaniphila; transferase LHPSNNSFVLCGVDYFRQAIAYVRKKRPDARFFFFSDDMEWVRENLWMEDAVYVEHTELMPDYMDLYLM Para- [Para- TLCRGHIISNSTFSFWGAYLAVDGNGMKIYPRRWFRDPTWITPPIFSEEWVGL prevotella prevotella xylaniphila xylaniphila] YIT 11841 Dethio- WP_ 491897177 glycosyl- 22.84 MFQYAFGRALALDLGLDLKLDISNFGSDSRPFSLGIYSLTKNIPFGCYLSTSTRLKVMTKKLRRWGVWGMD 248 sulfovibrio 005658864.1 transferase KNMPGLVEPFPPVLVSLDEVLSEKLSHLFVDGYWQSEKYFSRYSDVIRSDFRVIEESSAFLAWKKRMLSEPG peptido- family 11 GSISVHVRRGDYVTDSSANRVHGVLPIEYYLRAKEILNTISDGLVFYVFTDDPVWARNNLCLGDKTIYVSGEDL vorans; [Dethio- KDYEELALMSCCDHHVVANSSFSWWGAWLGQDTSTVTIAPGRWFRKMDSSFVIPDNWIKIWT Dethio- sulfovibrio sulfovibrio peptido- peptido- vorans] vorans DSM 11002 Lachno- WP_ 510896192 protein 22.83 MIIIQVMGGLGNQLQQYALYRKFVRMGKEQRLDISWFLDKEKRGEVLAERELELDYFDRLIYETCTPEEKEQLI 249 spiraceae 016229292.1 [Lachno- GSEGVAGKLKRKFLPGRIRWFHESKIYHPELLQMENMYLSGYFACEKYYADILYDLREKIQFPVNDHPKNIKM bacterium spiraceae AQEMQERESVSVHLRRGDYLDEKNTAMFGNICTDAYYCKAIEYMKTLCSKPHFYIFSDDIPYVRQRFTGEEYT 10-1 bacterium VVDINHGRDSFFDMWLMSRCRHNICANSTFSFWGARLNSNDNKIMIRPTIHKNSQVFVKEEMEQLWPG 10-1] WKFISPDGGIK Treponema WP_ 513872223 protein 22.82 MFCAAFVEALKHAGQKVFVDTSLYNKGTVRSGIDFCHNGLETEHLFGIKFDEADKADVHRLSTSAEGLLNRIR 250 maltophilum; 016525279.1 [Treponema RKYFTKKTHYIDTVFRYTPEVLSDKSDRYLEGFWQTEKYFLPIESDIRTLFRFRQPLSEKSAAVQSALQAQEPAS Treponema maltophilum] LSASIHVRRGDFLHTKTLNVCTETYYNNAIEYAAKKYAVSAFYVFSDDIQWCREHLNFFGARSVFIDWNIGAD maltophilum SWQDMVLMSMCRCNIIANSSFSWWAAWLNAASDKIVLAPAIWNRRQLEYADRYYGYDSDIPETWIRIP ATCC 51939 I Bacteroides WP_ 511022363 protein 22.79 MKLVSFTAGLGNQLFQYCFYRYLLNKFPNEKIYGYYNKKWLKKHGGIIIEHFFDVKLPRSTRWINLYGQYLRIIY 251 massiliensis; 016276676.1 [Bacteroides KCFSCGVSKDDDFEMNRTMFVGYWQDQCFFSGINISYKKNLVISEKNTWLLGEILKCNSVAIHFRRGDYMLP Bacteroides massiliensis] QFKKIFGEVCTVKYYLKSIRKVEEKISEPVFFVFSDDIDWVKQNFTFNKVYFVDWNKGQNSFWDMYLMSQC massiliensis SANIIANSTFSFWGAYLNKNNPFVIYPQKWVRTNLKQPNIFTKTWMAL dnLKV3 Enterococcus YP_ 389869137 family 11 22.71 MTVLTLGGGLGNQMFQYGYARYIQKIHREKFIYINDSEVIKEADRFNSLGNLNTVNIKVLPRIISKPLNETERLV 252 faecium; 006376560.1 glycosyl- RKIMVRLFGVAGFNESAIFQSLNKFGIYYHPSVYKFYESLKTGFPIKIIEGGFQSWKYLETCPEIKQELRVKYEP Enterococcus transferase MGENLRLLNLISQSESVCVHIRRGDYLSPKYKHLNVCDYQYYFESMNYIISKLNNPTFFIFSNTSDDLDWIKEN faecium DO; [Enterococcus YSLPGKIVYVKNDNPDYEELRLMYSCKHFILLSNSTFSWWAQYLSNNSGIVIAPEIWNRLNHDGIADLYMPNW Enterococcus faecium DO] ITMKVNR faecium EnGen0035 Bacteroides; WP_ 490442319 glycosyl- 22.67 MDVVVIFNGLGNQMSQYAYYLAKKKVNPNTKVIFDIMSKHNHGYDLERAFGIEVNKTLLIKVLQIIYVLSRK 253 Bacteroides 004313284.1 transferase FRLFKSVGVRTIYEPLNYDYTPLLMQKGPWGINYYVGGWHSEKNFMNVPDEVKKAFMFREQPNEDRFNE sp. 2_1_22; family 11 WLQVIRGDNSSVSVHIRRGDYMNIEPTGYYQLNGVATLDYYHEAIDYIRQYVTPHFYVFSNDLDWCKEQF Bacteroides [Bacteroides] GVENFFYIECNQGVNSWRDMYLMSECHYHINANSTFSWWGAWLCKFEDSITVCPERFIRNVVTKDFYPER sp. 2_2_4; WHKIKSC Bacteroides sp. D1; Bacteroides xylanisolvens SD CC 2a; Bactertoides xylanisolvens SD CC 1b; Bacteroides ovatus CAG:22 Synecho- YP_ 326781960 glycosyl- 22.6 MIGFNALGRMGRLANQMFQYASLKGIARNTGVDFCVPYHEEAVNDGIGNMLRTEIFDSFDLQVNVGLLNK 254 coccus phage 004322362.1 transferase GHAPVVQERFFHFDEELFRMCPDHVDIRGYFQTEKYFKHIEDEIREDFTFKDEILNPCKEMIAGVDNPLALHV S-SM2 family 11 RRTDYVTNSANHPPCTLEYYEALLKHFDDDRNVIVFSDDPAWCKEQELFSDDRFMISENEDNRIDLCLMSLC [Synecho- DDFIIANSTYSWWGAWLSANKDKKVIAPVQWFGTGYTKDHKTSKLIPDGWTRIATA coccus phage S-SM2] Geobacter YP_ 404496189 glycosyl- 22.58 MDIHVLSYGLGNQLSYAQFFINRRQLMQRAYAFYAFKQHNGYELDRIFGLKEGLPWYLQFVRVVFRLGISRR 255 metalli- 006720295.1 transfer FYSKRTADFVLSLFRIKVIDEAYNYEFDPSLLKPWFGIRILYGGWHDSRYFHPSEAAVRTAFSFPPLDDVNDAIL reducens; [Geobacter QQIDAVYGVSIHVRRGDYLKGINSNLFGGIATLEYYRNAIGWAITYCKHRSLEIKFYVFSDDIDWCKQNLGLR Geobacter metalli- DAVYVSGNSKTDSEKDILLMSHCRANIIANSTFSWWAAWLNQQPNKVVICPTKFINTDSPNQTIYPAAWH metalli- reducens  QIEG reducens GS- GS-15] 15; Geobacter metalli- reducens RCH3 Lachno- WP_ 551037245 protein 22.58 MIIVRFHGGLGNQMFEYAFYRYMTNKYGADNVIGDMTWFDRNYSEHQGYELKKVFDIDIPAIDYKTLAKIH 256 spiraceae 022780989.1 [Lachno- EYYPRYHRFAGLRYLSRMYAKYKNKHLKPTGEYIMDFGPSQYIHNDAFDKLDTNKDYYIEGVFCSDAYIKYYE bacterium spiraceae NQIKKDLTFKPNYSQHTKDMLPKIEETNSVAIHVRRGDYVGNVFDIVTPDYYRQAVNYIRERVENPVFFVSD NK4A136 bacterium DMDYIKANFDFLGDFVPVHNCGKDSFQDMYLISRCRHMIIANSSFSYFGALLGEKDSTIVIAPKKYKADEDLA NK4A136] LARENWVLL Bacteroides WP_ 495419937 protein 22.56 MGFIVNMACGLANRMFQYSYYLFLKKQGYKVTVDFYRSAKLAHEKVAWNSIFPYAEIKQASRLKVFLWGGG 257 coprophilus; 008144634.1 [Bacteroides SDLCSKVRRRYFPSSTNVRTTTGAFDASLPANTARNEYIIGVFLNASIVEAVDDEIKKCFTFLPFTDEMNLRLKK Bacteroides coprophilus] EIEECESVAIHVRKGKDYQSRIWYQNTSCMEYYRKAILQMKEKLQHSKFYVFTDNVDWVKENFQEIDYTLVE coprophilus GNPADGYGSHFDMQLMSLCKHNIISNSTYSWWSAFLNRNPEKVVIAPEIWFNPDSCDEFRSDRALCKGWI DSM 18228 = VL JCM 13818 Bacteroidetes; WP_ 495895157 alpha-1,2- 22.53 MKIVCLKGGLGNQMFEYCRFRDLMESGHDEVYLFYDHRRLKQHNGLRLSDCFELELPSCPWGIKLVVWGLK 258 Capnocytophaga 008619736.1 fucosyl- ICRAVGVLKRLYDDEKPEAVLIDDYSQHRRFIPNARRFFFRQFLAELQSGFVQMIRAVDYPVSVHVRRGDYL sp. oral transferase HPSNSSFGLCGVDYFQQAIAYVRKKRPDARFFFFSDDMEWVRENLWMEDAVYVEHTELLPDYVDLYLMTL taxon 329 str. [Bacter- CRGHHIISNSTFSFWGAYLAVDGNGMKIYPRRWFRDPTWTSPPIFSEEWVGL F0087 oidetes] Para- prevotella clara YIT 11840 Butyrivibrio WP_ 551026242 glycosyl- 22.47 MLIIQIAGGLGNQMQQYAVYTKLREMGDKVKLDLSWFDPQVQKNMLAPREFELPIFGGTDYEECSDAYERD 259 sp. NC2007 022770361.1 transferase ALLKQGAFAAIAGKVLKKLGLRDEANPKVFSEKEMYHPEVFELEDKYIKGYFACQKYYGDIMDKLQEKFIFPE [Butyrivibrio HSDPDLHARNMALVERMEREPSVSVHIRRGDYLDPSNVEILGNIATEQYYQGAMDYFTVKEPDTHFYIFTSD sp. NC2007] HEYAREKFSDESKYTIVDWNNGKNSVQDLMLMSHCKHNICANSTFSFWGARLNKRPDKTVIRTYKMRNN QPVNPQIMHDYWKGWILMDEKGSII Para- WP_ 495903957 glycosyl- 22.45 MKIILVFTGGLGNQMFAYAFYLYLKRLFPQERFYGLYGKKLSEHYGLEIDKWFKVSLPRQPWWVLPVTGLFYL 260 prevotella 008628536.1 transferase YKQCVPNSKWLDLNQEICKNPRAVIFFPFKFTKKYIPDDNIWLEWKVDESGLSEKNRLLLSEIRSSDCCFVHVR xylaniphila; [Para- RGDYLSPTFKSLFEGCCTLSYYQRALKSMKEISPFVKVFCFSDDIQWVKQNLELGNRAVFVWNSGTDSPLD Para- prevotella MYLMSQCRYGIMANSTFSYWGARLGRKKKRIYYPQKWWNHGTGLPDIFPNTWVKI prevotella xylaniphila] xylaniphila YIT 11841 Blautia WP_ 492742598 protein 22.44 HEIHVYLTGRLGNQLFQYAFARHLQKEYGGKIICNIYELEHRSEKAAWVPGKFNYEMSNYKLNDSILIEDIKLP 261 hydro- 00594476.1 [Blautia] WFADFSNPIIRIVKKVIPRIYFNLMASKGYLLWQKNSYINIPAIRNNEIIVNGWWQDVRFFHDVEAELSNEIVP genotrophica TTKPISENEYLYNIAERENSVCVSIRGGNYLVPKVKKKLFVCDKEYFYNAIELLIKSKVRNAIFVFSDDLEWVKSYI DSM 10507; KLEEKFPECKFYYESGKDTVEEKLRMMTKCKHFIISNSSFSWWAQYLAKNENFIVIAPDAWFTNGDKNGLYI Blautia; DDWILIPTQTKKM Blautia  hydro- genotrophica CAG:147 Geobacter YP_ 189425804 glycoside 22.44 MITVLLNGGLGNQLFQYAAGRALAEKHDVELLLDLSRLQHPKPGDTPRCFELAPFNIKASLLAEEGRQPLGGSY 262 lovleyi; 001952981.1 hydrolase QACMHRLLLKASIPLWGSIILKEQGCGFDPLIFRAPSCILDGFWQDECYFKQITSLLQQELSLKAPSPALRKAS Geobacter family SVLSDATVAVHVRRGDYVTNPAAASFHGICSQDYYQAAVANILTSYPDSQFLVFSDDPAWCQEHLDLGQPF lovleyi SZ protein RLAADFGLNGSAEELVLISRCAHQIIANSSFSWWGAWLNPSPHKLVVAPCRWFTPAITTNDLLPETWVRLP [Geobacter lovleyi SZ] Lachno- WP_ 551037435 protein 22.41 MVISHLSGGFGNQLYSYAFAYAVAKARKEELWIDTAIQDAPWFFRNPDILNLNIKYDKRVSYKIGEKKIDKIFN 263 spiraceae 022781176.1 [Lachno- RINFRNAIGWNTKIINESDMPNIDDWFDTCVNQKGNIYIKGNWSYEKLFISVKQEIIDMFTFKNELSKEANDI bacterium spiraceae AQDINSQETSVHIHYRLGDYVKIGINIVPDYFIASAMTSMVEKYGNPVFYSFSEDNDWVKKQFEGLPYNIKYVE NK4A136 bacterium YSSDDKGLEDFRLYSMCKHQIASNSSYSWWGAYLNNNPNKYIIAPTDYNGGWKSEIYPKHWDVRPFEFLK NK4A136] Bacteroides WP_ 492426440 glycosyl- 22.37 MFHYKFLLFGGGLGNQIFEYYFYLWLRKKYPNIVFLGCYRKASFKAHNGLEISDVFDVDLPNDGGLSGRFISYV 264 vulgatus; 005840359.1 transferase LSVLSRIIPSLSMKANTEYSSKYLLINAYQPNLLFYLNEEKIKFRPFKLDEVNRRLLNSIKMESSVSIHVRRGDYLF Bacteroides family 11 GQYRDIYSNICTLAYYQKAVDKCKGILESPRFFVFSDDIEWARDVFVGREYEFVSNNIGKNSFIDMFLMSNCKI vulgatus [Bacteroides QIIANSTFSYWAAYLSNSLVKIYPAKWINGIERPNIFPDNWIGL PC510 vulgatus] Planctomyces YP_ 325110698 glycosyl- 22.37 MIIARIENGLGNQLFKYAAGRALSKHRTSLYTIPGSVRKPHETFILSKYFNVQAKSVSPFLLQTGFRLRLLKGYE 265 brasiliensis; 004271766.1 transferase NHSFGFDPRFETTRNNTVVSGNFQSARYFLPFFDQINRELTLKPEVVDGLESVYPHVLESLRTPNSVVNHIRLG Planctomyces family protein DYVSSGYDICGPEYYAKAISRLQQLGHELRAFVFSDTPQAASRFLPADIDAQIMSEFPEVRDAARSLTVERSTI brasiliensis [Planctomyces RDYFLMQQCRHFVIPSNNFSYWAALLSSSDGDVIYPNRWYIDIDTSPRDLGLAPAEWTPIPLT DSM 5305 brasiliensis DSM 5305] Butyrivibrio WP_ 551028648 glycosyl 22.36 MIILQIAGGLGNQMQQYALRKLLKCGKTVKLDLSWFGPEIQKNMLAPFEFELVLFDLPFEICTKEEFDALIK 266 sp. AE2015 022772730.1 transferase QNLFQKIAGKVSQKLGKSASSNAKVFVETMYHEEIFDLDDVYITGYFACQYYYDDVMAELQDLFVFPSHSIP [Butyrivibrio ELDQRNAVLASKMEKENSVSVHIRRGDYLSPENVGILGNIASDKYYESAMNYFLEKDENTHFYIFTNDHEYAR sp. AE2015] EHYSDESRYTIIDWNTGKNSLQDLMLMSHCKGNICANSTFSWGARLNKRPDRELVRTLKMRNNQEAQPEI MHEYWKNWILIDENGVIV Roseovarius WP_ 497499658 alpha-1,2- 22.34 MTDTPPPSQVITSRLFGGAGNQLFQYAAGRALADRLGCDLMIDARYVAGSRDGDCFTHFAKARLRRDVA 267 nubinhibens 009813856.1 fucosyl- LPPAKSDGPLYRALWRKFGRSPRFHRERGLGVDPEFFNLPRGTYHGYWQSEQYFGPDTDALRRDLTLTTAL ISM; transferase DAPNAAMAAQIDAAPCPVSFHVRRGDYIAAGAYAACTPDYYRAAADHLATTLGKPLTCFIFSNDPAWARD Roseovarius [Roseovarius NLDLGQDQVIVDLNDEATGHFDMALMARCAHHVIANSTFSWWGAWLNPDPDKLVVAPRNWFATQALH nubinhibens nubinhibens] NPDLIPEQWHRL Eubacterium WP_ 548315094 protein 22.33 MIEVNIVGQLGNQMFEYACARQLQKKYGGEIVLNTYEMRKETPNKFLSILDYKSENDVKIISDKPLSSANANN 268 sp. CAG:581 022505071.1 [Eubacterium YLVKIMRQYFPNWYFNFMAKRGTFVWKSARKYKELPELNEQLSKHIVLNGYWQCDKYFNDVVDTIREDFTP sp. CAG:581] KYPLKAENEQQLEKIKSTESVSVTIRRGDFMNEKNKDTFYICDDDYFNKALSKIKELCPDCTFGFSDDVWEIKK NVNFPGEVYFESGNDPVWEKLRLMSACKHFVLSNSSFSWWAQYLSDNNNKIVVAPDIWYKTDGPKKTALY QDGWNLIHIGD Providencia AFH02807.1 383289327 glycosyl- 22.26 MKINGKESSMKIKQKKIISHLIGGLGNQLFQYATSYALAKENNAKIVIDDRLFKKYKHGGYRLDKLNIIGEKISS 269 alcalifaciens transferase IDKLLFPLILCKLSQKENFIFKSTKKFILEKKTSSFKYLTFSDKEHTKMLIGYWQNAIYFQKYFSELKEMFVPLDIS [Providencia QEQLDLSIQHAQQSVALHVRRGDYISNKNALAMHGICSIDYYKNSIQHINAKLEKPFFYIFSNDKLWCEENLT alcalifaciens] PLFDGNFHIVENNSQEIDLWLISQCQHHIIANSTFSWWGAWLANSDSQIVITPDPWFNKEIPIPSPVLSHWL KLKK Salmonella AFW04804.1 411146173 glycosyl- 22.26 MFSCLSGGLGNQMFQYSAAYILKKNICHAQLIIDDSYFYCQPQKDTPRNFEINQFNIVFDRVTTDEEKRAISKL 270 enterica transferase RKFKKIPLPLFKSNVITEFLFGKSLLTDEDFYKVLKKNQFTVKMNACLFSLYQDSSLINKYRDLILPLFTINDELLQ [Salmonella VCQQLDSYGFICEHTNTTSLHIRRGDYVTNPHAAKGHGTLSMNYYSQAMNYVDHKLGKQLFIIFSDDVQWA enterica] AEKFGGRSDCYIVNNVNCQFSAIDMYLMSLCNNNIIANSTYSWWGAWLNKSEEVLVIAPRKWFAEDKESLL AVNDWISI Sulfuro- YP_ 268680398 protein 22.18 MIIIKIMGGLASQLHKYSVGRALSLKYNTELKLDIFWFDNISGSGTIREYHLDKYNVVAKIATEQEIKQFKPNKY 271 spirillum 003304829.1 Sdel_1779 LLKINNLFQKFTNWKINYRNYCNESFISLENFNLLPDNIYVEGEWSDRYFSHIKEILQKELTLKSEYMDSTNHF deleyianum; [Sulfuro- LAKQSSDFAHDDNASKLHCTCSLEYYKKALQYISKNLLKMKLLIFSDDLDWLKPNFNFLDNVEFEFVEGFQDY Sulfuro- spirillum EEFHLMTLSKHNIIANSGFSLFFAWLNINHNKIIISLSEWVFEEKLNKYIIDNIKDKNILFLENLE spirillum deleyianum deleyianum DSM 6946] DSM 6946 Pseudo- YP_ 374329930 alpha-1,2- 22.15 MSVASQVRISGAARRRKLKPTLIVRIRGGIGNQLFQYALGRKIALETGMKLRFDRSEYDQYFNRSYCLNLFKT 272 vibrio sp. 005080114.1 fucosyl- QGLSATESEMSAVLWPAQSFGQTVKLCRKFYPFYQRRYIREDELLQDSETPVLKQSAYLDGYWQTWEIPFSI FO-BEG1 transferase MEQLRDEITLKKPMVLERLKLLQRIKSGPSAALHVRYGDYSQAHNLQNFGLCSAGYYKGAMDFLTERVPGLT [Pseudo- FYVFSDSPERAREVVPQQENVYFSDPMQDGKDHEDLMVMSSCDHIVTANSTFSWWAAFLNGNEDKHVIA vibrio sp. PLKWFKNPNLDDLIVPPHWQRL FO-BEG1] Prevotella WP_ 496529942 alpha-1,2- 22.11 MKIVCIKGGLGNQLFEYCRYHGLLRQHNNHGVYLHYDRRRTKQHGGVWLDKAFLITLPTEPWRVKLMVM 273 sp. oral 009236633.1 fucosyl- ALKMLRKLHLFKRLYREDDPRAVLIDDYSQHKQFITNAAEILNFRPFAQLDYVDEITSEPFAVSVHVRRGDYLL taxon 472 str. transferase PANKANFGVCSVHYYLSAAVAVRERHPDARFFVFSDDIEWAKMNLNLNLPNCVFVEHAQPQPDHADLYLMSL F0295; [Prevotella CKGHIIANSTFSFWGAYLSMGSSAIAIYPKQWFAEFTWNAPDIFLGHWIAL Prevotella sp. oral sp. oral taxon 472] taxon 472  Butyrivibrio WP_ 551008155 glycosyl- 22.08 MLIIRVAGGLGNQMQQYAMYRKLKSLGKEVKLDLSWFDVENQEGQLAPRKCELKYFDGVDFEECTDAERA 274 fibrisolvens 022752732.1 transferase YFTKRSILTKALNKVFPATCKIFEETEMFHPEIYSFKDKYLEFYFLCNKYYDDILPFIQNEIVPKHSDPKMQKRN [Butyrivibrio EELMERMDGWHTASIHLRRGDYITEPQNEALFGNIATDAYYDAAIRYVLDKDYQTHFYIFSNDPEYAREHYS fibrisolvens] DESRYTIVTGNDGDNSLLDMELMSHCRYNICANSTFSFWGARLNKRSDKEMIRTFKMRNNQEVTAREMTD YWKDWILIDEKGNRIF Lewinella WP_ 522059857 protein 22.04 MVISRLHSGLGNQMFQYAFARRIQLQLNVKLRIDLSILLDSRPPDGYIKREYDLDIFKLSPAYHCNPTSLRILYA 275 persica 020571066.1 [Lewinella PGKYRWSQVVRDLARKGYPVYMEKSFSVDNTLLDSPPDNVIYQGYWQSERYFSEVANTIRKDFAFQHSIQP persica] QSESLAREIRKEDSVCLNIRRKDYLASPTHNVTDETYYENCIQQMRERFSGARFFLFSDDLVWCREFFAHFHD VVIVGHDHAGPKFGNYLQLMAQCHHYIIPNSTFAWWAAWLGERTGSVIMAPERWFGTDEFGYRDVVPER WLKVPN

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

What is claimed is:
 1. A method for producing a fucosylated lacto-N-fucopentaose I (LNF I) oligosaccharide in a bacterium comprising providing a bacterium comprising an exogenous lactose-utilizing α(1,2) fucosyltransferase enzyme, wherein said α(1,2) fucosyltransferase enzyme has at least 90% sequence identity to amino acid sequence SEQ ID NO: 13; and culturing said bacterium in the presence of lactose, wherein said LNF I oligosaccharide is produced, wherein said bacterium further comprises β1,3-N-acetylglucosaminyltransferase and β1,3-galactosyltransferase.
 2. The method of claim 1, wherein said α(1,2) fucosyltransferase enzyme comprises Methanosphaerula palustries FutR.
 3. The method of claim 1, further comprising retrieving the fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
 4. The method of claim 1, wherein said bacterium further produces 2′-fucosyllactose (2′-FL), lactodifucotetraose (LDFT), or lacto-N-difucohexaose I (LDFH I).
 5. The method of claim 1, wherein the bacterium further comprises an exogenous lactose-utilizing α(1,3) fucosyltransferase enzyme and/or an exogenous lactose-utilizing α(1,4) fucosyltransferase enzyme.
 6. The method of claim 5, wherein the exogenous lactose-utilizing α(1,3) fucosyltransferase enzyme comprises a Helicobacter pylori 26695 futA gene.
 7. The method of claim 5, wherein the exogenous lactose-utilizing α(1,4) fucosyltransferase enzyme comprises a Helicobacter pylori UA948 FucTa gene or a Helicobacter pylori strain DMS6709 FucT III gene.
 8. The method of claim 1, wherein said bacterium further comprises a reduced level of β-galactosidase activity, a defective colanic acid synthesis pathway, an inactivated adenosine-5′-triphosphate (ATP)-dependent intracellular protease, or an inactivated endogenous lacA gene, or any combination thereof.
 9. The method of claim 8, wherein said method further comprises culturing said bacterium in the presence of tryptophan and in the absence of thymidine.
 10. The method of claim 8, wherein said reduced level of β-galactosidase activity comprises a deleted or inactivated endogenous lacZ gene and/or a deleted or inactivated endogenous lacI gene of said bacterium.
 11. The method of claim 10, wherein said reduced level of β-galactosidase activity further comprises an exogenous lacZ gene, wherein said exogenous lacZ gene comprises an β-galactosidase activity level less than wild-type bacterium.
 12. The method of claim 8, wherein said reduced level of β-galactosidase activity comprises an activity level less than wild-type bacterium.
 13. The method of claim 12, wherein said reduced level of β-galactosidase activity comprises less than 6,000 Miller units of β-galactosidase activity.
 14. The method of claim 12, wherein said reduced level of β-galactosidase activity comprises less than 1,000 Miller units of β-galactosidase activity.
 15. The method of claim 8, wherein said bacterium comprises a lack gene promoter immediately upstream of a lacY gene.
 16. The method of claim 8, wherein said defective colanic acid synthesis pathway comprises the inactivation of a wcaJ gene of said bacterium is deleted.
 17. The method of claim 8, wherein said inactivated ATP-dependent intracellular protease is a null mutation, inactivating mutation, or deletion of an endogenous lon gene.
 18. The method of claim 17, wherein said inactivating mutation of an endogenous lon gene comprises the insertion of a functional E. coli lacZ⁺ gene.
 19. The method of claim 8, wherein said bacterium further comprises a functional lactose permease gene.
 20. The method of claim 19, wherein said bacterium comprises E. coli lacY.
 21. The method of claim 8, wherein said bacterium further comprises an exogenous E. coli rcsA or E. coli rcsB gene.
 22. The method of claim 8, wherein said bacterium further comprises a mutation in a thyA gene.
 23. The method of claim 8, wherein said bacterium accumulates intracellular lactose in the presence of exogenous lactose.
 24. The method of claim 8 wherein said bacterium accumulates intracellular GDP-fucose.
 25. The method of claim 1, wherein said bacterium is E. coli.
 26. The method of claim 1, wherein said production strain is a member of the Bacillus, Pantoea, Lactobacillus, Lactococcus, Streptococcus, Proprionibacterium, Enterococcus, Bifidobacterium, Sporolactobacillus, Micromomospora, Micrococcus, Rhodococcus, or Pseudomonas genus.
 27. The method of claim 1, wherein said production strain is selected from the group consisting of Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus thermophiles, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans, Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, Xanthomonas campestris Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, Lactococcus lactis, Streptococcus thermophiles, Proprionibacterium freudenreichii, Enterococcus faecium, Enterococcus thermophiles), Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Pseudomonas fluorescens and Pseudomonas aeruginosa.
 28. The method of claim 1, wherein said bacterium comprises a nucleic acid construct comprising an isolated nucleic acid encoding said α(1,2) fucosyltransferase enzyme.
 29. The method of claim 28, wherein said nucleic acid is operably linked to one or more heterologous control sequences that direct the production of the enzyme in the bacterium.
 30. The method of claim 29, wherein said heterologous control sequence comprises a bacterial promoter and operator, a bacterial ribosome binding site, a bacterial transcriptional terminator, or a plasmid selectable marker.
 31. The method of claim 1, wherein the amino acid sequence of said enzyme comprises the amino acid sequence of FutR (SEQ ID NO: 13). 